Cytokine Activation Leads to Acidification and Increases
Maturation of Mycobacterium avium-Containing Phagosomes in
Ulrich E. Schaible,2* Sheila Sturgill-Koszycki,* Paul H. Schlesinger,†and David G. Russell3*
Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with lysosomes, do not acidify
due to a paucity of vacuolar proton-ATPases, yet remain accessible to recycling endosomes. During the course of mycobacterial
infections, IFN-?-mediated activation of host and bystander macrophages is a key mechanism in the regulation of bacterial
growth. Here we demonstrate that in keeping with earlier studies, cytokine activation of host macrophages leads to a decrease in
MAC viability, demonstrable by bacterial esterase staining with fluorescein diacetate as well as colony-forming unit counts from
infected cells. Analysis of the pH of MAC phagosomes demonstrated that the vacuoles in activated macrophages equilibrate to pH
5.2, in contrast to pH 6.3 in resting phagocytes. Biochemical analysis of MAC phagosomes from both resting and activated
macrophages confirmed that the lower intraphagosomal pH correlated with an increased accumulation of proton-ATPases. Fur-
thermore, the lower pH is reflected in the transition of MAC phagosomes to a point no longer accessible to transferrin, a marker
of the recycling endosomal system. These alterations parallel the coalescence of bacterial vacuoles from individual bacilli in single
vacuoles to communal vacuoles with multiple bacilli. These data demonstrate that bacteriostatic and bactericidal activities of
activated macrophages are concomitant with alterations in the physiology of the mycobacterial phagosome.
Immunology, 1998, 160: 1290–1296.
acteristics that support the intracellular survival and growth of
these pathogens in professional phagocytes. Previous studies es-
tablished that phagosomes containing MAC4and Mycobacterium
tuberculosis have restricted fusigenicity with lysosomes (1–6) and
do not acidify (2, 7, 8) due to a block in accumulation of vacuolar
proton-ATPase (2). Despite the apparent sequestration of MAC
and M. tuberculosis vacuoles outside the endosomal/lysosomal
continuum, recent studies have shown that these vacuoles are rel-
atively dynamic, maintaining access to glycosphingolipids and
glycoconjugates (9) from the host cell plasmalemma.
Immunoelectron microscopical studies by Clemens and Horwitz
(4) on M. tuberculosis-infected MO revealed the presence of MHC
class II and transferrin receptor in mycobacteria-containing phago-
The Journal of
ollowing phagocytosis, Mycobacterium species reside and
multiply in phagosomes of the host’s macrophages (1).
Mycobacteria-containing phagosomes have unique char-
somes. Indeed, we demonstrated recently that MAC phagosomes,
although restricted in acquisition of proton-ATPases, have access
to cathepsins B, L, and D which enter phagosomes early in their
maturation (10). In spite of this, the high phagosomal pH limits
processing and activation of cathepsin D. The hypothesis that my-
cobacterial phagosomes represent early endosomes stabilized in
this stage was given further credence in two studies showing their
accessibility to transferrin, a marker for the recycling endosomal
system (10, 11).
Several independent studies published during the past 10 years
have highlighted the role of certain cytokines in mycobacterial
infections. It is accepted that activation of MO by T cell-, NK cell-,
or macrophage-derived cytokines such as IFN-?, IL-1, granulo-
cyte-macrophage-CSF, and TNF-?, alone or in concert, can con-
tribute to the antimycobacterial potential of these cells, resulting in
control of the infection in vitro (12–19) and in vivo (20, 21). Here,
we have studied the influence of MO activation by IFN-? and LPS
on the maturation of MAC phagosomes and correlated these
changes with mycobacterial survival. The data presented suggest
that MAC phagosomes are shifted from an early to a late endoso-
mal stage of phagosome maturation by MO activation, which is
concomitant with a reduction in mycobacterial growth and
Materials and Methods
The following Abs were used in this study: the mAb ID4B against
LAMP-1 was obtained from the Developmental Hybridoma Bank, Iowa
City, IA; mouse mAbs E11 and H9 against the vacuolar proton-ATPase E
subunits were generous gifts from Dr. S. Gluck (Washington University,
St. Louis, MO); rat mAb against transferrin receptor (R17/18, Tib217)
were obtained from American Type Culture Collection, mouse mAb anti-
digoxigenin was purchased from Boehringer Mannheim, Indianapolis, IN.
The rabbit polyclonal Ab to cathepsin D was a generous gift from Dr. S.
Kornfeld (Washington University). Secondary species-specific Abs labeled
*Departments of Molecular Microbiology and†Physiology and Cell Biology, Wash-
ington University, School of Medicine, St. Louis, MO 63110
Received for publication May 21, 1997. Accepted for publication October 9, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was in part supported by a postdoctoral fellowship of the stipend program
for infectious diseases by the Deutsche Bundesministerium fu ¨r Bildung, Wissen-
schaft, Forschung, und Technologie, Germany (U.E.S.) and by Grants AI33348 and
HL55936 (D.G.R.). D.G.R. is a recipient of the Burroughs Wellcome Scholar award
in Molecular Parasitology.
2Current address: MPI fur Infektionsbiologie, Monbijoustrasse 2, D-10117 Berlin,
3Address correspondence and reprint requests to Dr. David G. Russell, Department
of Molecular Microbiology, Washington University School of Medicine, 660 S. Eu-
clid Avenue, St. Louis, MO 63110. E-mail address: email@example.com
4Abbreviations used in this paper: MAC, Mycobacterium avium complex; LAMP 1,
lysosome-associated membrane protein 1; NHS, N-hydroxysuccinimide; MO, bone
marrow-derived macrophages; ?2m, ?2-macroglobulin; NO, nitric oxide; iNOS, in-
ducible nitric oxide synthase.
Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00
with horseradish peroxidase were purchased from Jackson Immunore-
search Laboratories, West Grove, PA. Iron-loaded transferrin and human
?2m were both purchased from Calbiochem, San Diego, CA.
Bone marrow-derived MO and activation
MO were differentiated and maintained in culture as described previously
(3, 9). MO were grown in bacteriologic petri dishes and split by placing
them in cold PBS for 30 min followed by gentle scraping. After splitting,
MO were allowed to establish a monolayer for at least 3 days in cell culture
flasks before use. MO were activated according to the following proce-
dures: For early time points (2–4 h), macrophages were incubated for 16 h
with 400 U/ml recombinant mouse IFN-? and 200 to 500 ng/ml LPS for 2 h
before infection. For late time points (5 d), macrophages were incubated
with 400 U/ml recombinant mouse IFN-? and 200 to 500 ng/ml LPS added
on day 4 postinfection.
Bacteria, infection, colony-forming units
MAC 101 from frozen stocks derived from the first passage following
isolation from a mouse was cultured in Middlebrook broth (Difco, Detroit,
MI) and used within 3 days of thawing. Translucent colony appearance as
an indication of virulence was tested before each experiment and routinely
revealed ?1% opaque (avirulent) colonies. MO cultures were infected with
MAC 101 in a 10:1 ratio in DMEM without antibiotics supplemented with
5% L929 conditioned medium, 5% horse serum for 2 h, washed twice, and
cultured for the time period indicated for each experiment.
MO cultures (1 ? 104) were set up in 12-well tissue culture plates in
duplicate and infected with MAC in a bacteria-MO ratio of 10:1. CFUs
were determined as follows. The infected MO monolayers were lysed in 1
ml of PBS containing 0.5% Nonidet P-40, and lysates were passaged seven
times through a 25-gauge tuberculin needle. The lysates were diluted in
Middlebrook broth (1/100; 1/1000, 1/10,000), and 100-?l aliquots were
plated in duplicate onto Middlebrook agar plates and incubated for 10 days.
Viability stain for mycobacteria
Following the protocol of McDonough and Kress (22), infected MO were
incubated in DMEM/10% FCS containing 4 mg/ml carboxyfluorescein di-
acetate (Molecular Probes, Eugene, OR) for 10 min at 37°C, fixed in 4%
formaldehyde, and counterstained with Evans blue (Sigma Chemical Co.,
St. Louis, MO). Mycobacteria-infected MO were examined under an epi-
fluorescence microscope and scored blind for strong (metabolically active)
or low/no fluorescence (metabolically inactive).
MAC labeled with NHS-carboxyfluorescein (Boehringer Mannheim)
were used to infect MO on quartz glass. The fluorescence of the total
cell population was measured at different time points fluorometrically
and compared with a standard pH curve using NHS-carboxyfluorescein-
labeled MAC in suspension and in nigericin-treated MO, as described
previously (2, 23).
Transferrin was labeled covalently with NHS-digoxigenin (Molecular
Probes) at a 10:1 molar excess in PBS, pH 7.8, for 30 min on ice and
purified over Kwiksep exocellulose columns (Pierce Chemical Co., Rock-
ford, IL). Human ?2m was labeled with
Heights, IL) by Iodobeads (Pierce Chemical Co.) according to the manu-
125I (Amersham, Arlington
Phagosomes containing MAC were isolated according to a protocol de-
scribed earlier (9, 10). Contamination of phagosomes with other cellular
material or digoxigenin-transferrin was evaluated for each experimental
preparation by a crossover method described (9, 10). In brief, bacilli were
added to unlabeled macrophages that were scraped and combined with an
equal number of macrophages that were either labeled metabolically with
[35S]methionine or incubated in digoxigenin transferrin. The level of con-
tamination was 3 to 10% for metabolically labeled macrophages and below
the level of detection for digoxigenin-transferrin macrophages.
PAGE and Western blot
Isolated phagosomes were lysed in 3? SDS buffer, boiled, and separated
by SDS-PAGE (12%) under reducing conditions. After blotting onto ni-
trocellulose, blots were blocked in PBS containing 0.05% Triton X-114,
0.05% Tween-20, 10% goat serum, and 5% milk powder; incubated in the
respective Abs; and developed using the Lumiglo system (Pierce
Macrophages activated before or postinfection were fixed in 2% glutaral-
dehyde in PBS, osmicated, dehydrated through ethanol, and embedded in
Spurr’s resin. Thin sections were cut, contrasted with uranyl acetate and
Reynold’s lead, and examined in a Jeol 100CX electron microscope. The
distribution of bacteria/vacuoles were scored by examining 200 vacuoles
for each condition. No more than 6 vacuoles were scored per cell.
Effects of MO activation on MAC viability
To study the effect of MO activation on MAC infection in vitro, we
performed infection experiments on both resting and activated mu-
rine MO. Preliminary experiments confirmed previous reports that
activation with IFN-? (400 U/ml) alone postinfection did not in-
duce a strong microbicidal response or the marked alteration in
vacuole physiology detailed below. Full mycobactericidal activity
and iNOS expression requires a second signal, TNF-?, which can
be induced by LPS (24, 25). We therefore added LPS at 200 ng/ml
in concert with IFN-? to maximize MO stimulation. Cell mono-
layers of MO were infected in a ratio of 10 bacilli/MO which
infected ?90% of the cells. MO were cultivated in medium plus or
minus IFN-?, and LPS was added before infection or 4 days
postinfection (Fig. 1). In resting MO cultures, the number of re-
coverable CFU showed a modest decline over the first 4 days
until the numbers could be seen to be increasing from 5 days
postinfection. In contrast, in activated MO, this decline was
more pronounced and sustained until the macrophage mono-
layer started to disintegrate at 6 days postinfection. Despite the
ability of activated macrophages to regulate the bacterial pop-
ulation, bacterial death was neither rapid nor efficient, and CFU
analysis indicated that many bacteria persisted within activated
The initial stages of infection were characterized by static bac-
terial numbers, even in resting macrophages. This bacterial popu-
lation is likely to be extremely heterogeneous with respect to vi-
ability. To reduce the heterogeneity of the bacterial population
under study, we commenced analysis with addition of IFN-? and
LPS 4 days postinfection when the bacteria were entering expo-
nential growth phase (Fig. 1). In nonactivated MOs, CFUs in-
creased from 4 days postinfection and stayed at high levels until 10
days postinfection. In contrast, the CFUs from MO cultures acti-
vated 4 days postinfection peaked 2 days after activation and
slowly decreased until 6 days after activation (10 days postinfec-
tion) (Fig. 1). During this same time, bacteria in resting MO had
entered into exponential growth. To further characterize the effect
of MO activation on MAC viability, we used a viability stain
method based on bacterial esterase activity (22). The proportion of
metabolically active (esterase-positive) mycobacteria increased
during the entire observation period in resting MO; whereas in MO
activated 4 days postinfection, the number of metabolically active
mycobacteria stayed low and decreased until 6 days postactivation
(10 days postinfection) (Fig. 1b).
Although the trend is comparable, the viability stain method
“overestimated” the relative number of live bacilli in activated
macrophages relative to the CFU data. This discrepancy may re-
flect bacterial aggregation on isolation from activated macro-
phages before plating or persistence of esterase activity in non-
replicative bacteria. Despite this variation, both data sets argue
that activation with IFN-? and LPS renders murine bone mar-
row-derived MO capable of controlling an established MAC
1291The Journal of Immunology
MAC are unable to block phagosome acidification in activated
In resting macrophages, phagosomes containing inert particles, or
other pathogens like Leishmania, rapidly acidify from the extra-
cellular pH to below pH 5.0 (2). In contrast, the pH within phago-
somes containing MAC shows a more restricted drop and equili-
brates to pH 6.2 (2). In this study, we compared the
intraphagosomal pH of MAC phagosomes in resting vs activated
MO. Resting MO were compared with MO activated for 16 h with
IFN-? and for 2 h with LPS before infection. Carboxyfluorescein-
labeled MAC were bound to the MO on ice, unbound bacteria were
washed off, and the MOs were placed at 37°C for the time periods
stipulated. As detailed previously (2), the pH of MAC phagosomes
in resting MO did not drop below pH 6.2 (Fig. 2). In contrast, in
activated MO, the pH in the MAC-containing phagosomes
dropped to pH 5.2 within 180 min of internalization (Fig. 2). There
was an intriguing “rebound” in the pH at 90 to 120 min postin-
fection which was observed in three independent experiments.
Acidification of MAC phagosomes correlates with acquisition of
We had attributed the restricted acidification observed in myco-
bacteria phagosomes to the paucity of vacuolar proton-ATPases in
this compartment (2). To test whether MO activation reverses this
phenotype, similar numbers of phagosomes were isolated from
resting MO or from MO activated with IFN-? for 16 h and LPS for
2 h, separated by SDS-PAGE, blotted, and probed for the E subunit
of the vacuolar proton-ATPase. At both 4 h (Fig. 3) and 5 days
(Fig. 4) postinfection, minimal proton-ATPase could be detected
in phagosomes from resting MO. In contrast, MAC phagosomes
from activated MO infected for the same periods of time contained
significant amounts of proton-ATPase (Figs. 3 and 4). Phagosome
preparations were normalized for protein content (10); however, to
demonstrate that comparable amounts of protein were loaded in the
lanes, blots were subsequently probed for LAMP-1 (Figs. 3 and 4).
(b starts on day 4, at time of activation). The experiments reveal a modest drop in viability of infecting bacilli before their entry into logarithmic growth
phase in resting macrophages at days 4 to 5 postinfection. The bacilli in activated macrophages never enter into this growth phase, although they do persist
in low numbers. a, MO were infected with MAC 101 and cultured in the presence or absence of activating cytokines added before or after infection (400
U/ml IFN-? added 16 h before infection and 200 ng/ml LPS added 2 h before infection or 400 U/ml IFN-? and 200 ng/ml LPS added 4 days postinfection).
Data represent the mean from CFUs from wells plated in duplicate; the SD is calculated on the two independent data sets. b, MO seeded onto coverslips
and activated on day 4 as described above. At time points indicated, coverslips were stained with carboxyfluorescein diacetate for 10 min at 37°C, fixed,
and counterstained, and green fluorescent vs total mycobacteria were counted. Data of percentages of metabolically active mycobacteria represent the
mean ? SD as counted in six microscopic fields of two coverslips for each time point. Similar data were obtained in four independent CFU assays and
two independent viability staining experiments.
Influence of MO activation on MAC viability as revealed by colony counts (a) and in situ staining (b) for metabolically active mycobacteria
in resting MO or MO activated for 16 h with 400 U/ml IFN-? and 2 h with
200 ng/ml LPS before infection. These data demonstrate that the limited
acidification of MAC vacuoles in resting macrophages is reversed by in-
cubation of MO in macrophage-activating cytokines. MO were seeded onto
quartz glass chips and infected with MAC labeled with NHS-carboxyfluo-
rescein. pH-dependent alteration of fluorescence was determined spec-
trofluorometrically during the time range indicated and compared against a
pH standard of carboxyfluorescein-labeled MAC as outlined in Materials
and Methods. Within the time frame of the experiment, the majority of the
fluorescent label remained associated with the surface of the bacilli. Data
represent the mean of measurements from three independent experiments
for each time point.
Fluorometric determination of the pH of MAC phagosomes
1292 PHAGOSOME MODULATION IN ACTIVATED M. AVIUM-INFECTED MACROPHAGES
MAC vacuoles in activated macrophages are no longer
accessible to transferrin
Previous work from Clemens and Horwitz (4, 11), de Chastellier et
al. (5), and our laboratory (9, 10) had suggested that mycobacterial
phagosomes maintain communication with the early or recycling
endosomal system of their host cell. This was demonstrated by the
characterization of the flux of transferrin through MAC vacuoles
even in 9-day-old infections (10). If MAC vacuoles in activated
MO undergo a functional translocation to a later endosomal stage,
this should be mirrored by a loss of accessibility to transferrin. To
test this, both resting and activated MO infected with MAC 5 days
previously were incubated with digoxigenin-labeled transferrin for
45 min, washed intensively, and lysed. Comparable numbers of
phagosomes from resting vs activated MO were separated by SDS-
PAGE, blotted, and probed for digoxigenin. MO lysates revealed
that similar amounts of digoxigenin-transferrin were taken up by
both populations of MO (Fig. 4c). Also, as described previously,
transferrin was readily detected in MAC phagosomes from resting
MO. In contrast, only minimal amounts of digoxigenin-transferrin
were present in MAC phagosomes from activated MO (Fig. 4c).
These were the same phagosome preparations probed for proton-
ATPase (E subunit) and LAMP 1 as discussed. These data suggest
that in activated MO, MAC-containing phagosomes are shifted
functionally toward a late stage in endosomal maturation charac-
terized by accumulation of proton-ATPases and the loss of inter-
section with transferrin-carrying vesicles. Additional experiments
were performed using125I-labeled ?2m, which usually proceeds
along the lysosomal pathway following endosomal uptake. As ex-
pected, only small amounts of ?2m could be detected in isolated
MAC phagosomes from resting MO, whereas up to four times
more ?2m was found in phagosomes from activated MO (data not
Alterations in phagosome physiology appears to precede the
drop in MAC viability
It is still unclear whether the increased maturation of MAC phago-
somes is symptomatic of or causal to the loss of bacterial viability.
The complex responses of macrophages to cytokine activation ren-
der this question difficult to resolve. Furthermore, there are no data
regarding the effects of macrophage activation on the regulation of
phagosome/lysosome fusion independent
However, electron microscopic analysis of infected MO shortly
after activation indicates that one of the first phenotypic alterations
in activated macrophages is the coalescence of individual M.
avium-containing vacuoles into communal vacuoles with many ba-
cilli (Figs. 5 and 6). This is observed in macrophages activated
before infection and in macrophages activated 4 days postinfection
and examined 5 days postinfection. Quantitation of the distribution
of bacilli in vacuoles (Fig. 6) demonstrates that at early time
points, 2 h postinfection, there is a marked decrease in the numbers
of bacilli in individual vacuoles in activated vs resting MO. Fur-
thermore, the more established the infection, such as 5-day infec-
tions in resting MO, the higher is the proportion of single bacteria
in individual vacuoles. However, at both the 2-h and 5-day time
points, the majority of bacilli show few signs of damage or deg-
radation, even in the communal vacuoles of activated MO. These
data provide a preliminary indication that the merging of vacuoles
precedes any marked drop in bacterial viability, as assessed by
CFUs shown in Figure 1.
This study details experiments designed to examine the effects of
MO activation on the biology of MAC-containing vacuoles and
describes data that correlate the alterations in vacuole physiology
with the subsequent development of mycobacteriostatic and my-
cobactericidal properties of the infected host MO.
The role of cytokine-activated MO in the modulation of myco-
bacterial infections shows qualitative variation with mouse strain
and bacterial species but has been shown to be critical in protection
through experiments conducted on IFN-? and TNF-? receptor
4 h postinfection from resting MO (lane 1) and MO activated for 16 h with
400 U/ml IFN-? and 2 h with 200 ng/ml LPS (lane 2) before infection.
Nitrocellulose membranes were probed with anti-LAMP 1 (a) and anti-
proton ATPase E subunit (b) Abs. There was a marked increase in the
amount of proton-ATPase present in phagosomes isolated from activated
MO. This correlates with the pH drop observed in Figure 2. Phagosomes
were isolated 4 h postinfection and normalized for protein content before
SDS-PAGE. Comparable results were obtained in six independent
Immunoblot of M. avium-containing phagosomes isolated
and 4) isolated 5 days postinfection from resting MO (lanes 1 and 3) and
MO activated for 16 h with 400 U/ml IFN-? and 200 ng/ml LPS (lanes 2
and 4) before phagosome isolation. The membranes were probed with
anti-LAMP 1 (a), anti-proton ATPase E subunit (b), and anti-digoxigenin
transferrin (c). The amount of proton-ATPase is increased in MAC phago-
somes from activated MO relative to the amount of LAMP-1. In contrast,
digoxigenin-transferrin was readily detectable in mycobacterial phago-
somes from resting MO but was absent from phagosomes from activated
MO. Lanes 1 and 2 were run with total macrophage homogenate from the
same preparations of resting (lane 1) and activated (lane 2) MO from
which the phagosomes were isolated. Phagosomes were isolated 5 days
postinfection and normalized for protein content before SDS-PAGE. Com-
parable results were obtained in more than six independent experiments.
Immunoblot of M. avium-containing phagosomes (lanes 3
1293The Journal of Immunology
knockout mice (26, 27). In vitro, IFN-?- and/or TNF-?-activated
murine MO are able to inhibit growth of Mycobacterium bovis and
M. tuberculosis (12, 15–17, 26). Similarly, Appelberg and Orme
(18) showed bacteriostasis by IFN-?-activated murine MO toward
some but not all MAC isolates tested. The levels of MO-derived
TNF-? produced by infected MO varied with bacterial isolate and
appeared to be crucially involved in the protective response (18,
19, 27). In systems where mycobactericidal behavior has been in-
duced, there is some debate as to the mechanism(s) involved.
Growth inhibition of M. tuberculosis and M. bovis by activated
MO has been attributed to the release of NO but not to reactivated
oxygen metabolites (16). In contrast, growth inhibition of MAC by
activation of MO with IFN-? and LPS. a, Resting MO 2 h following infection. The bacilli tend to be sequestered in individual vacuoles that show little
evidence of lysosomal fusion. b, Activated MO 2 h following infection. The bacteria are observed more frequently in communal vacuoles that contain dense,
lysosomal matrix. MO were activated with IFN-? (400 U/ml) for 16 h and LPS (500 ng/ml) for 2 h before infection. c, Resting MO 5 days postinfection.
M. avium persist and divide in individual vacuoles. Many of these replicating organisms have prominent ribosomes (arrowed). d, Activated MO 5 days
postinfection. Again, there is a marked tendency for the bacteria to be in vacuoles containing multiple bacilli. Although there is little obvious degeneration
of the bacilli, the ribosomes are not as numerous or developed as those seen in c. MO were activated on day 4 with IFN-? (400 U/ml) for 16 h and LPS
(500 ng/ml) for 2 h before processing. Comparable results were observed in two independent experiments.
Electron micrographs of murine bone marrow-derived MO infected with M. avium, revealing alterations in vacuole morphology following
1294PHAGOSOME MODULATION IN ACTIVATED M. AVIUM-INFECTED MACROPHAGES
activated MO has been suggested to be independent of NO (18)
and mediated by superoxide production (19), possibly in the con-
text of enhanced phagosome/lysosome fusion (18).
Despite the body of data demonstrating the central role of iNOS
in the regulation of murine mycobacterial infections and other in-
tracellular pathogens, the evidence is all of the same type, either
protection through the use of inhibitors such as nonhydrolyzable
arginine analogues or the use of iNOS knockout mice (28–30).
Although these experiments all indicate that iNOS is necessary for
protection, they do not shed light on whether it is sufficient. Ob-
viously, iNOS fulfills a necessary function in the regulation of
many infections, but a full appreciation of its mode of action must
take into account the cascade of other physiologic changes that
occur during macrophage activation.
The maintenance of mycobacterial vacuoles within the early en-
dosomal machinery appears to require metabolic activity because
dead bacilli are internalized into vacuoles that acidify and fuse
with lysosomes (11) (S. Sturgill-Koszycki et al., unpublished ob-
servations). It is therefore important to appreciate which event
comes first: the death, or compromise of the infecting microbe; or
the differentiation of their compartment into an acidic, hydrolyti-
cally competent lysosome. If the latter is true, this translocation
could drastically alter both the environment and cofactors that
would potentiate the efficacy of NO. Our data on murine macro-
phages infected with Mycobacterium avium indicate that activation
facilitates acidification of mycobacterial vacuoles in both de novo
and established infections. The functional translocation toward
more lysosomal compartments appears to precede any marked
drop in microbial viability, suggesting that it is the product of an
alteration in macrophage physiology, rather than a consequence of
The lysosomal environment of activated macrophages could po-
tentiate NO toxicity in several ways (24, 31). Oxidation of NO to
nitrite and nitrate will be retarded at acid pH. NO can combine
with H2O2, the production of which is up-regulated in activated
macrophages, to make peroxynitrite (ONOO?). NO can release
metal ions, such as Fe2?, from metalloproteins which can combine
with H2O2to produce ?OH and hypervalent iron. Furthermore, the
activity of lysosomal hydrolases on the microbial cell wall likely
exposes more targets to oxidative attack. The microbicidal re-
sponses of activated macrophages are probably based on the com-
plex interactions of several antimicrobial phenomena, and more
work is required on the effects of activation on the regulation of
intracellular fusion within the endosomal/lysosomal continuum be-
fore these interactions can be appreciated.
Macrophage activation may also influence the availability or
accessibility of nutrients; e.g., retention within the recycling en-
dosomal machinery facilitates access to nutrients such as iron. M.
tuberculosis possess high affinity iron-binding proteins, and it has
been suggested that these are necessary because iron is a vital
factor for mycobacterial growth (32). This hypothesis is given fur-
ther credence by the report that the anti-MAC activity in human
serum is due to transferrin-mediated iron depletion and that apo-
transferrin can reduce intracellular growth of MAC (33, 34).
Results detailed in this study indicate that the loss of mycobac-
terial viability is a gradual process which appears subsequent to the
functional translocation of mycobacterial vacuoles to later endo-
somal and lysosomal compartments. This transition transfers my-
cobacteria from a relatively nonhostile environment and renders
them accessible to low pH, reducing conditions, acid hydrolases,
toxic peptides, and the potentiated effects of O and NO radicals at
low pH (33, 35). Under these conditions, many of the bacilli ap-
pear static but viable. How long this condition could persist is
unclear but it may mirror dormant M. tuberculosis infections in
vivo before reactivation (36). A fuller appreciation of the complex
interrelationships active during killing of Mycobacterium awaits
elucidation of the mechanism(s) whereby Mycobacterium spp. ar-
rest endosomal maturation and an understanding of the regulation
of endosomal/lysosomal fusion in activated macrophages.
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individual or communal vacuoles in resting vs activated MO at 2 h or 5
days postinfection, as detailed in Figure 5. There is a marked trend from
individual to communal vacuoles with activation of the host cells. More-
over, even in resting MO, there is an increase in individual vacuoles as the
bacteria enter exponential growth, suggesting that the “health” of the in-
fection is reflected in the percentage of vacuoles with single bacteria. Ac-
tivated MO cultures were incubated in IFN-? (400 U/ml) for 16 h and LPS
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1296 PHAGOSOME MODULATION IN ACTIVATED M. AVIUM-INFECTED MACROPHAGES