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Localized Reactive Oxygen and Nitrogen
Intermediates Inhibit Escape of Listeria
monocytogenes from Vacuoles in Activated
Macrophages
Jesse T. Myers, Albert W. Tsang and Joel A. Swanson
J Immunol 2003; 171:5447-5453; ;
doi: 10.4049/jimmunol.171.10.5447
http://www.jimmunol.org/content/171/10/5447
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References
The Journal of Immunology
Localized Reactive Oxygen and Nitrogen Intermediates Inhibit
Escape of Listeria monocytogenes from Vacuoles in Activated
Macrophages1
Jesse T. Myers,*† Albert W. Tsang,† and Joel A. Swanson2*†
A
critical stage in the life history and pathogenesis of Listeria monocytogenes (Lm)3 occurs just after it enters a
macrophage by phagocytosis. During a successful infection, Lm perforates the membranous vacuole that contains it and
escapes into the macrophage cytoplasm. There it can grow, divide,
and eventually nucleate host cell actin in a process that facilitates
transfer to neighboring cells (1). However, if the macrophage is
activated, by IFN-␥, bacterial products, and other cytokines produced during the immune response to infection, then escape from
vacuoles is inhibited, and bacteria are killed (2– 4).
Various molecules have been implicated in the listericidal activities of activated macrophages, but their relative effects on escape and killing have not been defined. Chief among these are
reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), which figure in defense against numerous pathogens (5–9). Activated macrophages produce NO by inducible NO
synthase (iNOS) encoded by the NOS2 gene, and ROI by the
NADPH oxidase complex. The GTPase Rab5a has been implicated
in listericidal activity, possibly via regulation of Rac2 and assem*Cellular and Molecular Biology Graduate Program and †Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109
Received for publication June 27, 2003. Accepted for publication September
12, 2003.
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.
1
bly of the oxidase complex (10, 11). Both ROI and RNI contribute
to murine resistance to Lm infection and to the listericidal activities
of activated macrophages (2, 12–16). However, it is not known
whether ROI and RNI affect escape from the vacuole or subsequent microbicidal functions.
The present studies examined the contributions of ROI and RNI
to Lm retention in vacuoles. The timing of Lm escape from vacuoles was measured, then gp91phox⫺/⫺ and NOS2⫺/⫺ knockout
mice, which are unable to generate superoxide and NO, respectively (17, 18), were used to define the relative contributions of
ROI and RNI to inhibition of escape in activated macrophages.
Finally, fluorescent methods were used to measure the timing of
ROI and RNI generation in vacuoles. These studies demonstrate
that escape occurs within the first 30 min after entry, and that the
combined actions of ROI and RNI inhibit Lm escape from vacuoles in activated macrophages.
Materials and Methods
Bacterial preparation
Lm strains 10403S and DP-L2161 (gifts from D. Portnoy, University of
California, Berkeley, CA) were maintained on brain-heart infusion agar
plates. For experiments, one or two bacterial colonies were added to 5 ml
of brain-heart infusion broth, shaken overnight at room temperature, diluted 1/6 the following morning, and shaken at 37°C for 1.5 h to obtain an
OD600 of 0.500. Bacteria were washed by pelleting and resuspending in
Ringer’s buffer (155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
2 mM NaH2PO4, 10 mM HEPES, and 10 mM glucose, pH 7.2) three times
before addition to macrophages.
This work was supported by grants from the National Institutes of Health.
2
Address correspondence and reprint requests to Dr. Joel A. Swanson, Department of
Microbiology and Immunology, University of Michigan Medical School, Ann Arbor,
MI 48109-0620. E-mail address: [email protected]
3
Abbreviations used in this paper: Lm, Listeria monocytogenes; BFA1, bafilomycin
A1; DAPI, 4⬘,6-diamidino-2-phenylindole; DHFF-BSA, dihydro-2⬘,4,5,6,7,7⬘hexafluorofluorescein coupled to BSA; DPI, diphenyleneiodonium; FeTPPS,
5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III); ␣-IL-10, IL-10-neutralizing antibody; iNOS, inducible NO synthase; L-NMMA, NG-monomethyl-L-arginine; MOI, multiplicity of infection; NOS, NO synthase; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; SOD, superoxide dismutase; TR,
Texas Red; LLO, listeriolysin.
Copyright © 2003 by The American Association of Immunologists, Inc.
Macrophages
Female NOS2⫺/⫺, gp91phox⫺/⫺, and homozygous wild-type (C57BL/6)
mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
NOS2⫺/⫺ and gp91phox⫺/⫺ mice had been backcrossed for at least 10 generations onto a C57BL/6 background. Bone marrow-derived macrophages
were cultured as previously described (19). After 5–9 days of growth, cells
were replated into 6-, 24-, or 96-well tissue culture dishes overnight in
DMEM plus 10% heat-inactivated FBS, 100 U/ml penicillin, and 100
␮g/ml streptomycin (Life Technologies, Gaithersburg, MD). Macrophages
were activated as previously described (20). Briefly, IFN-␥ (100 U/ml;
0022-1767/03/$02.00
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Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm.
Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of
reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm
escaped from vacuoles of nonactivated macrophages within 30 min of infection. Macrophages activated with IFN-␥, LPS, IL-6, and
a neutralizing Ab against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar
escape to varying degrees. Measurements of Lm escape in macrophages from gp91phoxⴚ/ⴚ and NO synthase 2ⴚ/ⴚ mice showed that
vacuolar retention required ROI and was augmented by RNI. Live cell imaging with the fluorogenic probe dihydro-2ⴕ,4,5,6,7,7ⴕhexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized
to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were
generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the localization of ROI and
RNI to vacuoles, and by their combined actions in a small space The Journal of Immunology, 2003, 171: 5447–5453.
5448
R&D Systems, Minneapolis, MN), LPS (100 ng/ml; List Biological,
Campbell, CA), and a neutralizing Ab against IL-10 (␣-IL-10, 5 ␮g/ml;
R&D Systems) were included in the overnight incubation medium, followed by the presence of ␣-IL-10 and 5 ng/ml IL-6 (Calbiochem, San
Diego, CA) during the course of the experiment.
Vacuole escape assay
Imaging with dihydro-2⬘,4,5,6,7,7⬘-hexafluorofluorescein
covalently linked to BSA (DHFF-BSA)
Macrophages were plated onto 25-mm circular coverslips (2.5 ⫻ 105/coverslip) overnight and then mounted in a temperature-controlled stage at
37°C, mounted on an inverted microscope (TE-300; Nikon, Tokyo, Japan)
equipped with a cooled CCD camera (Quantix; Photometrics, Tuscon, AZ),
filter wheel (␭ 10 –2; Sutter Instruments, Novato, CA), and a phase contrast
⫻100 oil objective (N.A.1.4). Cells were pulsed for 5 min with Lm (MOI,
⬃1) along with DHFF-BSA (1 mg/ml; Molecular Probes). TR-dextran
(m.w., 10,000; 0.1 mg/ml) was included in the pulse to verify uptake of the
nonfluorescent DHFF-BSA into phagosomes. Coverslips were then washed
three times with 5 ml of Ringer’s buffer. MetaMorph software (Universal
Imaging, Downington, PA) was used to create macros that sequentially
acquired phase contrast and fluorescence images exciting with 485 nm
(F485) and 580 nm light (F580) using a multichroic beam-splitter (Omega
Optical, Brattleboro, VT). Once an Lm-containing vacuole was located,
images were acquired every 30 s for ⬃10 min to assemble time-lapse
sequences. Otherwise, coverslips were scanned over a period of 25 min,
using phase contrast microscopy to locate vacuoles containing Lm. Phase
contrast and fluorescence images of each vacuole were then acquired.
MetaMorph was used to quantitate fluorescent conversion of DHFFBSA. Regions were traced around phase contrast images of Lm-containing
phagosomes. The corresponding region was copied to the F485 and F580
images, and the average F485 and F580 pixel intensities of the region were
logged into a Excel spreadsheet (Microsoft, Redmond, WA) along with the
time the image was acquired. To calculate the average amount of conversion of DHFF-BSA (see Fig. 6C), the individual phagosomal fluorescence
intensities were pooled and averaged for each condition, and a background
value of 127 (average F485 in a phagosome without probe; calculated in a
separate experiment) was subtracted. Values were then displayed as a percentage of the response measured in wild-type activated macrophages.
For experiments comparing oxidation of DHFF-BSA with phagosomal
area (see Fig. 7), F485, F580, time, and pixel area were recorded for Lm
phagosomes of activated macrophages. Phagosomes were grouped into
three arbitrary size groupings representing small, medium, and large
phagosomes. The F485 intensity was divided by the F580 intensity to normalize for the amount of probe within the phagosome. Student’s t test
(2-tailed distribution) was used to calculate the statistical significance of
the differences between samples.
To compare sizes of Lm-containing vacuoles from activated and nonactivated macrophages, cells were plated on 25-mm circular coverslips
(3.0 ⫻ 105) overnight, then mounted on the temperature-controlled stage of
an inverted microscope and maintained at 37°C. Cells were pulsed for 5
min with 100 ␮l of Ringer’s buffer containing hemolysin-deficient Lm
mutant hly (strain DP-L2161) plus 1.0 mg/ml Lucifer Yellow and then
washed with 15 ml of Ringer’s buffer. Cells were excited with 436 nm of
light, and the coverslip was scanned for vacuoles containing Lm, which
were identified as fluorescent compartments with darker regions created by
dye displaced by bacteria. Phase contrast and fluorescence images (excitation, 436 nm; emigration, 480 nm) were acquired of each Lm-containing
vacuole. In MetaMorph, regions were drawn around the bacteria and the
vacuoles in which they were contained, and the pixel area of each vacuole
was logged into Excel.
Listericidal assay
Macrophages were plated onto 13-mm coverslips and incubated overnight
in medium alone (control) or medium plus one of the following combinations
of ingredients: 1) IFN-␥ and LPS, 2) IFN-␥, LPS, plus IL-6 added during the
experiment, or 3) IFN-␥, LPS, and ␣-IL-10, plus IL-6 added during the experiment. Macrophages were then incubated with Lm for 30 min, washed, and
incubated for the indicated times in medium with gentamicin (50 ␮g/ml) for
0.5–7.5 h, after which macrophages were fixed and stained with DAPI to
determine the number of bacteria per infected macrophage.
Results
Lm escapes from vacuoles within 30 min
To visualize the chemistries involved in the listericidal response of
activated macrophages, it was important to know the time frame in
which those chemistries would be active. A drop in pH is required
for Lm to escape from the vacuole into the cytosol (21). The proton
ATPase inhibitor BFA1 prevents endocytic vacuole acidification
and can be used to prevent phagosomal escape of Lm (22). Nonactivated macrophages were treated with BFA1 at various times
after infection with Lm. Bacteria that had escaped into the cytosol
were identified by their association with filamentous actin, as indicated by staining with TR-phalloidin. When cells were treated
with BFA1 immediately following infection, only ⬃20% of bacteria engulfed by macrophages managed to escape into the cytoplasm (Fig. 1). Later additions of BFA1 showed an increased
amount of escape that leveled off after 30 min, at which point
addition of BFA1 had no measurable effect on the escape of Lm.
Therefore, nearly all vacuolar escape occurred within 30 min of
infection. The mechanisms used by activated macrophages to retain Lm within the vacuole must be active during this brief period
following entry.
FIGURE 1. Lm escape from phagosomes within 30 min. Macrophages
were infected with Lm for 15 min, followed by incubation in medium and
gentamicin (25 ␮g/ml) for 2 h. BFA1 (500 nM) was added at the indicated
times after the infection. Cells were fixed and stained with DAPI and TRphalloidin. For each condition ⱖ50 infected macrophages were scored for
the presence of TR-phalloidin-labeled Lm. BFA1 inhibited bacterial escape
when it was added within the first 30 min of infection. Each data point
represents the mean ⫾ SEM of three experiments.
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Macrophages were plated onto 13-mm circular coverslips (7.5 ⫻ 104/coverslip) in a 24-well plate and activated overnight. Macrophages were
washed twice with Ringer’s buffer, then incubated 15 min at 37°C with Lm
in DMEM and 10% FBS without antibiotics (multiplicity of infection
(MOI), ⬃0.1). For experiments determining the timing of vacuole escape,
bafilomycin A1 (BFA1; Sigma-Aldrich, St. Louis, MO) was added to cells
at a concentration of 500 nM at various times after infection. Superoxide
dismutase (SOD; 150 U/ml; Sigma-Aldrich), catalase (1500 U/ml; SigmaAldrich), and 5,10,15, 20-tetrakis(4-sulfonatophenyl) porphyrinato iron
(III) (FeTPPS; 100 ␮M; Calbiochem, La Jolla, CA) were included during
the infection as noted. For experiments involving the use of NG-monomethyl-L-arginine (1 mM; L-NMMA; Calbiochem) or diphenyleneiodonium (DPI; 10 ␮M; Molecular Probes, Eugene, OR), cells were pretreated
with the inhibitor for 15 min. L-NMMA or DPI were then included in the
medium for the duration of the experiment. Following infection, cells were
washed four times with Ringer’s buffer and incubated in DMEM, 10%
FBS, and 25 ␮g/ml gentamicin for 3.5 h. Cells were then fixed for 15 min
at room temperature in cystoskeletal fix (30 mM HEPES, 10 mM EGTA,
0.5 mM EDTA, 5 mM MgSO4, 33 mM potassium acetate, 5% polyethylene
glycol 400, and 4% paraformaldehyde), followed by washing three times
with PBS and 2% goat serum and permeabilization with 0.3% Triton X-100
in PBS for 5 min. Permeabilized cells were then washed three times in PBS
and goat serum for 5 min each and incubated for 15 min in PBS and goat
serum with Texas Red-phalloidin (TR-phalloidin; 2 U/ml from 200 U/ml
stock in methanol; Molecular Probes) and 4⬘,6-diamidino-2-phenylindole
(DAPI; 2 ␮g/ml, Molecular Probes). Cells were washed three times for 5
min with PBS and goat serum and mounted on glass slides with Prolong
Antifade (Molecular Probes). For each coverslip, 50 macrophages with
DAPI-labeled bacteria were scored for colocalization of bacteria with filamentous actin.
LISTERICIDAL ACTIVITIES OF MACROPHAGE VACUOLES
The Journal of Immunology
5449
Activation of bone marrow-derived macrophages
ROI and RNI both inhibit vacuolar escape by Lm
The ability of activated macrophages to inhibit Lm escape was
used to develop assays for the microbicidal activities of ROI and
RNI. Macrophages were infected at a low MOI (⬃0.1), such that
each infected macrophage initially contained only one bacterium.
Two hours after infection, cells were fixed and stained with TRphalloidin, and the percentage of infected macrophages with actinpositive bacteria was determined. The effects of various RNI and
ROI inhibitors on the ability of Lm to escape from the vacuole into
the cytoplasm were tested (Fig. 3). Activation of macrophages
with IFN-␥, LPS, IL-6, and ␣-IL-10 reduced Lm access to cytoplasm. Pretreatment of activated macrophages with the NO synthase inhibitor L-NMMA led to a dramatic increase in the escape
of Lm from vacuoles, indicating a role for NO in the prevention of
Lm escape. Treatment of activated macrophages with the superoxide scavenger SOD produced a small, but significant ( p ⬎ 0.05),
increase in Lm escape. The hydrogen peroxide scavenger catalase,
alone or in combination with SOD, did not significantly increase
vacuolar escape, suggesting that hydrogen peroxide did not contribute to the retention of Lm in vacuoles of activated macro-
FIGURE 2. Effects of macrophage activation on growth of Lm. Bone
marrow-derived macrophages activated with IFN-␥, LPS, IL-6, and
␣-IL-10 were infected for 15 min with Lm, then washed and chased for the
indicated times in gentamicin (25 ␮g/ml). Listericidal activity was measured by counting the number of DAPI-labeled bacteria per infected macrophage. The data are the mean ⫾ SEM of three independent experiments.
FIGURE 3. The effects of ROI and RNI inhibitors on escape of Lm from
phagosomes. Cytoplasmic localization of Lm was determined by colocalization with TR-phalloidin. The percentage of infected macrophages with
TR-positive bacteria was recorded in nonactivated macrophages and macrophages treated with IFN-␥, LPS, IL-6, and ␣-IL-10 (I, L, 6, and ␣-10).
Macrophages were treated with various combinations of 1 mM L-NMMA,
150 U/ml SOD, and 1500 U/ml catalase as indicated. Activated macrophages inhibited escape, and treatment of activated macrophages with LNMMA, SOD, and/or catalase prevented that inhibitory activity to varying
degrees. Values represent the mean ⫾ SEM. (n ⱖ 3).
phages. Treatment of activated macrophages with L-NMMA, SOD,
and catalase together resulted in the greatest amount of escape
from the vacuole, similar to that observed in nonactivated
macrophages.
As a separate approach for analyzing the contributions of ROI
and RNI to inhibition of Lm escape, vacuolar escape was measured
in macrophages from NOS2⫺/⫺ and gp91phox⫺/⫺ mice, in which
elimination of RNI or ROI, respectively, was specific and complete (Fig. 4A). Lm escape in nonactivated macrophages from
NOS2⫺/⫺ and gp91phox⫺/⫺ mice was not significantly different
from that in wild-type macrophages. However, although activation
of wild-type macrophages greatly reduced vacuolar escape of Lm,
escape from activated gp91phox⫺/⫺ macrophages was only slightly
FIGURE 4. Enhanced phagosomal escape of Lm in gp91phox⫺/⫺ and
NOS2⫺/⫺ macrophages. The percentage of infected macrophages with TRpositive bacteria was determined in activated and nonactivated macrophages from wild-type, gp91phox⫺/⫺, and NOS2⫺/⫺ mice, and in activated
wild-type, gp91phox⫺/⫺, and NOS2⫺/⫺ macrophages treated with 1 mM
phox⫺/⫺
L-NMMA (A). Activated macrophages from gp91
mice did not inhibit
escape any more than nonactivated macrophages. Activated macrophages
from NOS2⫺/⫺ mice showed intermediate levels of escape inhibition. B, Effect
of phagosomal escape in macrophages treated with 100 ␮M FeTPPS. FeTPPS
reversed some of the inhibition, indicating a possible role for peroxynitrite in
blocking escape. Data represent the mean ⫾ SEM (n ⱖ 3).
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Overnight treatment of peritoneal macrophages with IFN-␥ is sufficient to control the growth of Lm (4). The same treatment of bone
marrow-derived macrophages, however, does not lead to as high a
degree of listericidal activity (23). Therefore, we sought to enhance activation of bone marrow-derived macrophages with additional factors to augment the standard IFN-␥ and LPS method of
activation. ␣-IL-10, a cytokine secreted by macrophages that
down-regulates activation (24), was included in an overnight activation medium along with IFN-␥ and LPS. IL-6 was also included during the infection because it has been shown to increase
the listericidal activity of macrophages when added at the start of
infection (25). Activation of macrophages with IFN-␥ and LPS
alone initially reduced the number of bacteria per macrophage, as
measured by staining infected cells with DAPI and counting the
number of bacteria per cell, but bacterial numbers increased over
8 h (Fig. 2). In contrast, macrophages activated with IFN-␥, LPS,
IL-6, and ␣-IL-10 controlled the growth and replication of bacteria for
the duration of the experiment. Because activation with this cocktail
of ingredients resulted in more efficient containment of Lm, this activation protocol was used for all additional experiments.
5450
Localization of oxidative activity using DHFF-BSA
To visualize the timing and localization of the oxidative chemistries generated within the Lm vacuole, live cell imaging was performed with the probe DHFF-BSA. This is a relatively nonfluorescent molecule conjugated to BSA that, when oxidized, becomes
highly fluorescent (27). Macrophages were incubated for 5 min
with Lm, DHFF-BSA, and TR-dextran, a fluorescent indicator of
the volume of fluid taken into vacuoles and phagosomes. Timelapse sequences of Lm vacuoles containing DHFF-BSA showed a
rapid conversion of the relatively nonfluorescent probe to a fluorescein-like molecule (Fig. 5, A and B). Dye conversion was restricted to vacuoles containing bacteria. Probe-loaded macropinosomes, identifiable by their labeling with TR-dextran, generally
exhibited little DHFF-BSA fluorescence, even in cells containing
vacuoles with very bright DHFF-BSA signals (Fig. 5, C–E). On
rare occasions, fluorescent vacuoles with no apparent bacteria were
observed; the fluorescence in these vacuoles was less than that in
those that contained bacteria. It was unclear whether these vacu-
FIGURE 5. Localization of phagosomal oxidative activity with DHFFBSA. Macrophages were infected for
5 min in the presence of DHFF-BSA
and TR-dextran. A time-lapse series
showing, at 30-s intervals, phase contrast (A) and fluorescence images (excitation, 485 nm; B) of a phagosome
containing Lm (arrowhead) and
DHFF-BSA. Arrowheads indicate the
bacterium in corresponding positions
in the images. Phase contrast (C) and
fluorescence images exciting at 485 nm
(D) and 580 nm (E) were taken of a
macrophage infected with Lm. The arrow shows an Lm-containing phagosome, and arrowheads show macropinosomes loaded with the probe. DHFFBSA conversion was restricted to the
Lm vacuole. Bar ⫽ 5 ␮m.
oles contained unseen factors, such as bacterial proteins, which
generated a response by the macrophage.
One concern was that repeated exposure to 485 nm light in
time-lapse sequences contributed to the oxidation of DHFF-BSA.
To circumvent this problem, data were collected by scanning the
coverslip using phase contrast optics to identify Lm vacuoles, then
taking only one set of images for each vacuole (phase contrast,
F485, F580). Quantitative data were plotted as a function of time
after infection, then analyzed as a population of vesicles.
Fluorescent conversion of the probe was detectable almost as
soon as images could be acquired (Fig. 6A). DPI, an inhibitor of
flavoproteins that prevents the generation of both ROI and RNI
(28, 29) completely abrogated fluorescent conversion of DHFFBSA in activated macrophages (Fig. 6B). Thus, chemistries restricted to the phagosomes were converting DHFF-BSA very soon
after vacuole formation.
These chemistries were quantified by measuring the fluorescence in digital images. The degree of DHFF-BSA conversion was
determined by calculating the average fluorescence intensity under
various conditions (Fig. 6C). Nonactivated macrophages were capable of generating fluorescent vacuoles, although their average
fluorescence intensity was less than that of Lm vacuoles in activated macrophages.
The specificity of DHFF-BSA oxidation was examined using
gp91phox⫺/⫺ and NOS2⫺/⫺ macrophages. Phagosomes from
gp91phox⫺/⫺ macrophages exhibited minimal dye conversion; their
average fluorescence was similar to that observed in macropinosomes and in Lm vacuoles of DPI-treated macrophages (Fig. 6C).
Thus, ROI were required for the oxidation of DHFF-BSA, and
RNI alone were not sufficient to oxidize DHFF-BSA. Phagosomes
from NOS2⫺/⫺ macrophages as well as macrophages treated with
L-NMMA demonstrated a reduced average phagosomal fluorescence compared with wild-type activated macrophages (Fig. 6C).
Hence, RNI were not essential, but enhanced the fluorescent conversion of the probe, indicating either that some dye conversion
resulted from oxidation by peroxynitrite or that NO somehow enhanced ROI generation.
Although most Lm vacuoles of activated macrophages inhibited
escape, a smaller percentage showed dye conversion. We hypothesized that although the oxidative chemistries affecting Lm escape
and dye conversion were qualitatively similar, their effects on Lm
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less than that from nonactivated gp91phox⫺/⫺ macrophages. These
results differed significantly from data obtained with the ROI scavengers, SOD and catalase (Fig. 3), indicating that they may not
have inhibited ROI completely. Activation of NOS2⫺/⫺ macrophages led to a partial reduction in Lm escape, similar to the effects
of L-NMMA on wild-type macrophages (Fig. 4A). L-NMMA did
not have additional effect on escape in NOS2⫺/⫺ macrophages.
Therefore, L-NMMA was a specific and effective inhibitor of
iNOS. To examine the role of peroxynitrite, a microbicidal molecule formed by the reaction of superoxide and NO, on Lm escape,
macrophages were treated with the peroxynitrite-specific scavenger FeTPPS (26) (Fig. 4B). FeTPPS had no effect on phagosomal
escape in nonactivated macrophages, but escape in activated macrophages treated with the scavenger was approximately double that
observed in control cells ( p ⬍ 0.01).
In summary, the Lm escape studies indicated significant contribution of both ROI and RNI to the inhibition of escape from activated macrophages. The inability of macrophages from
gp91phox⫺/⫺ mice to inhibit escape suggests that ROI are essential.
The substantial, albeit incomplete, inhibition in NOS2⫺/⫺ mice
indicated its secondary importance, and the partial inhibition by
FeTPPS indicated a role for peroxynitrite.
LISTERICIDAL ACTIVITIES OF MACROPHAGE VACUOLES
The Journal of Immunology
escape were more efficient than their ability to convert the dye.
Moreover, we speculated that smaller, more tightly fitting phagosomes would oxidize DHFF-BSA more efficiently, since, presumably, oxidative chemistries could be more concentrated in a
smaller volume. To determine whether fluorescent conversion of
DHFF-BSA was affected by vacuolar size, the data presented in
Fig. 6A were reexamined, measuring the area of Lm vacuoles along
with the intensity of DHFF-BSA and TR-dextran fluorescence.
Phagosomes were divided into three arbitrary size categories that
were typical of tight-fitting, medium, and spacious phagosomes
(Fig. 7, A–C). DHFF-BSA fluorescence was normalized for the
amount of probe in the phagosome by dividing the DHFF-BSA
fluorescence (F485, indicating conversion) by the TR fluorescence
(F580, indicating the volume internalized). The conversion of
DHFF-BSA was significantly greater in small, tight-fitting phagosomes (0 –200 pixels) than in large, spacious phagosomes (⬎400
pixels; p ⬍ 0.02). Medium-sized phagosomes (200 – 400 pixels)
also oxidized DHFF-BSA more efficiently than large phagosomes,
but this difference was less significant ( p ⬍ 0.07). No size difference was observed between Lm vacuoles of activated and nonactivated macrophages (our unpublished observations).
FIGURE 7. DHFF-BSA converted more efficiently in smaller phagosomes. Representative images of phagosomes, with areas of 132 (A), 264
(B), and 531 (C) pixels, respectively. Bar ⫽ 2 ␮m. Fluorescence was normalized for the amount of ingested dye by dividing of DHFF-BSA (F485)
by the intensity of TR-dextran fluorescence (F580). Normalized fluorescence was recorded for phagosomes of three different size groupings
(⫾SEM).
Discussion
Activated macrophages, which are critical components of the cellmediated immune response against Lm (30 –33), control the
growth and replication of Lm by retaining the bacteria within the
phagocytic vacuole (4). ROI and RNI are important microbicidal
mediators and contribute to the listericidal activity of macrophages. We show here that ROI and RNI directly affect the retention of Lm in vacuoles of activated macrophages.
The role of ROI in the inhibition of escape was characterized
using gp91phox⫺/⫺ macrophages as well as the ROI-scavenging
enzymes, SOD and catalase. Activation did not improve the ability
of gp91phox⫺/⫺ macrophages to retain Lm within vacuoles (Fig.
4A), thereby indicating a requirement for ROI in enhanced vacuolar retention by activated macrophages. ROI alone (L-NMMAtreated or NOS2⫺/⫺ macrophages) could also reduce Lm escape,
although this reduction was not as great as when RNI were also
present. Results obtained with SOD and catalase did not produce
a phenotype as pronounced as when gp91phox⫺/⫺ macrophages
were used. Acidification of the vacuolar compartment may have
decreased the activity of SOD and catalase. Alternately, the rapid,
NO-dependent conversion of superoxide to peroxynitrite, which
should occur more rapidly than the scavenging of superoxide by
SOD (34), may have consumed superoxide before it could be scavenged by SOD.
RNI also aided in the retention of Lm in vacuoles of activated
macrophages. Activated NOS2⫺/⫺ or L-NMMA-treated macrophages showed decreased Lm phagosomal escape, but these effects
were not as complete as in wild-type macrophages (Fig. 4A). RNI
alone were not sufficient to retain Lm within vacuoles, because
gp91phox⫺/⫺ macrophages, producing RNI, but not ROI, could not
inhibit escape after activation. The effects of the NO inhibitor LNMMA on the escape and oxidation of DHFF-BSA were similar
to the effects observed in NOS2⫺/⫺ macrophages (Figs. 4A and
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FIGURE 6. Rapid conversion of DHFF-BSA in Lm phagosomes. Images of phagosomes containing Lm, DHFF-BSA, and TR-dextran were
taken over a period of 25 min following a 5-min infection. The average
fluorescence intensity (excitation, 485 nm) was recorded in activated macrophages (A) and activated macrophages treated with 10 ␮M DPI (B). Each
dot represents the fluorescence intensity of an individual phagosome. Time
zero was defined as the beginning of the 5-min infection. The average
fluorescence intensity (excitation, 485 nm) of Lm phagosomes, relative to
wild-type activated macrophages, was determined in activated and nonactivated wild-type, gp91phox⫺/⫺, and NOS2⫺/⫺ macrophages as well as in
wild-type macrophages treated with either DPI (activated only) or
L-NMMA (C; ⫾SEM). Measurements were also taken of probe-loaded
macropinosomes in macrophages not exposed to Lm. Macropinosomes, Lm
vacuoles of DPI-treated wild-type macrophages, and macrophages from
gp91phox⫺/⫺ mice showed background levels of fluorescence. Limited, submaximal conversion was detected in Lm vacuoles of nonactivated macrophages and in activated NOS2⫺/⫺ macrophages.
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fluorescent conversion of DHFF-BSA were similar to those that
retained Lm within the vacuole, with the exception of NOS2⫺/⫺
and L-NMMA-treated macrophages. In that case, escape was at an
intermediate level between activated and nonactivated control
macrophages (Fig. 4A), whereas fluorescent conversion of DHFFBSA in those cells was at the same level as in nonactivated macrophages (Fig. 6C). This raises the possibility that there might be
a small ROI/RNI-independent contribution to vacuolar retention of
Lm that does not lead to fluorescent conversion of DHFF-BSA.
Overall, however, DHFF-BSA oxidation was a good indicator of
the chemistries that led to vacuolar retention of Lm and demonstrated a rapid generation of oxidative chemistries localized to Lm
phagosomes.
Many Lm vacuoles were not fluorescent or were only slightly
more fluorescent than background. This may have been a result of
loss of probe, via fusion with lysosomes, or leakage through pores
formed by the pore-forming toxin listeriolysin O (LLO). Also,
vacuoles that were only partially formed after the 5-min pulse
would have lost probe when the noninternalized bacteria were
washed off. Nevertheless, many nonconverting vacuoles must have
contained probe, since they were TR-dextran positive. These vacuoles may have been imaged before ROI/RNI were generated
within the vacuole. Another possibility is that the concentrations of
the oxidative chemistries required to retain Lm in the vacuole are
less than those concentrations required to convert DHFF-BSA. Accordingly, some Lm vacuoles will have had ROI/RNI levels sufficient to block Lm escape, but insufficient to convert DHFF-BSA.
Because measurement of oxidative activity with DHFF-BSA
was not strictly quantitative, it is difficult to compare the responses
we observed with measurements that have been made using other
methods. Presumably, cells with a more prodigious oxidative
burst, such as peritoneal macrophages, would show an increased
conversion of DHFF-BSA, although experiments comparing the
conversion of DHFF-BSA by different cell types and different activation states have not been performed.
The rapid and localized response of the oxidative burst within
Lm vacuoles suggests recognition of the bacteria by the macrophage and invites speculation as to how the signals are generated
for this response. The bacteria were not serum-opsonized, although
we cannot rule out that something secreted by the macrophages
opsonized them rapidly. In the absence of opsonization by Abs or
complement, pattern recognition receptors are likely to contribute
to the generation of an immune response. The most likely candidates for the start of the signaling cascade are Toll-like receptors
(41, 42). It remains to be seen whether oxidation of DHFF-BSA is
a result of Toll-like receptor signaling.
Interestingly, DHFF-BSA was oxidized more efficiently in
small, tight phagosomes than in large spacious ones. Antimicrobial
chemistries generated into a tight-fitting phagosome would presumably be more concentrated than if they were generated within
a larger, spacious phagosome. The average size of Lm-containing
vacuoles was similar in activated and nonactivated macrophages
(our unpublished observations), indicating that activated macrophages do not restrict phagosomal size to enhance the effectiveness
of their oxidative chemistries. Spacious phagosome formation by
Lm may be a defense mechanism that reduces the effectiveness of
ROI and RNI within the phagosome.
Although it is now clear that the combined actions of ROI and
RNI are necessary for retention of Lm in vacuoles, their mechanism of action remains unknown. Rapid ROI/RNI-mediated killing
could have led to the retention of Lm within vacuoles. Preliminary
experiments have shown no effect of activation on the survival of
hemolysin-deficient mutant hly Lm in macrophages (our unpublished observations), making it unlikely that ROI/RNI directly
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6C). However, preincubation of cells with L-NMMA for 15 min
was necessary for complete suppression of NO generation (our
unpublished observations).
Our data are consistent with a model in which two types of
chemistries contribute to retention of Lm in vacuoles: one mediated by ROI alone, and another ROI-mediated chemistry that is
also dependent on the presence of RNI. This is similar to the RNIdependent and RNI-independent listericidal activities observed by
Muller et al. (12). One mechanism by which RNI could have enhanced the activity of ROI was via the generation of peroxynitrite,
which is formed by the reaction of superoxide and NO. Peroxynitrite is highly reactive and microbicidal (34 –37). Evidence for peroxynitrite-mediated vacuolar retention was demonstrated by the
increased percentage of cytoplasmic Lm in activated macrophages
treated with FeTPPS, a peroxynitrite scavenger with little SODmimetic activity (26). Attempts to localize nitrotyrosine, a reaction
product of peroxynitrite chemistry, have not succeeded. It is also
possible, however, that RNI enhance ROI-mediated phagosomal
retention by peroxynitrite-independent means. Along with being a
microbicidal molecule, NO is also commonly employed as a signaling molecule. NO-mediated chemistries may have primed the
macrophage to deliver a more potent oxidative burst.
Although RNI appear to be a significant contributor to antilisterial activity in our system, the importance of NO and RNI in
human macrophages is unclear. Cytokines that lead to iNOS expression in murine macrophages result in limited and inconsistent
expression of iNOS in human mononuclear phagocytes derived
from blood. Inducible NOS expression has been documented,
however, in macrophages isolated from patients with infectious or
inflammatory diseases (38). Thus, iNOS expression appears to be
more tightly regulated in human macrophages, but this does not
preclude the possibility that human macrophages use RNI more
efficiently to control the growth of Lm.
If peroxynitrite has a role in the vacuolar retention of Lm, then
superoxide and NO must be concomitantly present in the vacuole
within 30 min of infection, before the bacteria had escaped from
the vacuole. Therefore, it was necessary to determine whether the
localization and timing of the generation of oxidative chemistries
within the Lm vacuole were consistent with the generation of peroxynitrite. An imaging method was developed, using the fluorogenic probe DHFF-BSA, to study the timing and localization of the
oxidative chemistries generated by macrophages into the Lm vacuole. While it is relatively common to measure the oxidative burst
with fluorogenic probes either extracellularly or throughout the
cytoplasm, to our knowledge, ROI/RNI have not previously been
measured within a bacterial phagosome, the location where oxidative chemistries are most likely to have an effect. Fluorescent
conversion of DHFF-BSA is reported to be a result of oxidation by
hydrogen peroxide (27). DHFF-BSA oxidation was ROI dependent, as judged by the lack of fluorescence generated by
gp91phox⫺/⫺ macrophages (Fig. 6C). RNI alone were insufficient
for probe oxidation, but fluorescent conversion of DHFF-BSA was
reduced in both NOS2⫺/⫺ and L-NMMA-treated macrophages, indicating that RNI contribute to the ROI-mediated oxidation of
DHFF-BSA. Because NO can diffuse across membranes, it was
unclear whether RNI-mediated conversion of DHFF-BSA was due
to a localized generation of RNI into phagocytic vacuoles or if
constitutive generation of NO throughout the cell produced levels
of RNI within vacuoles that were sufficient for probe oxidation.
Although the reactivity of DHFF-BSA with peroxynitrite has not
been previously examined, 2⬘,7⬘-dichlorofluorescein, a molecule
similar to DHFF, is readily oxidized by peroxynitrite (39, 40),
suggesting the RNI-mediated enhancement of probe oxidation
could be due to reaction with peroxynitrite. Conditions that led to
LISTERICIDAL ACTIVITIES OF MACROPHAGE VACUOLES
The Journal of Immunology
killed Lm in our system. Alternately, ROI/RNI may have inactivated the pore-forming toxin LLO, thereby preventing the escape
of Lm into the cytosol. ROI and RNI could act cooperatively (e.g.,
generating peroxynitrite) or independently to inactivate LLO or to
stimulate other antilisterial mechanisms. It is also possible that NO
enhances the function of the NADPH-oxidase complex, either by
direct chemical modification of component proteins or by acting as
a second messenger to stimulate other mechanisms that potentiate
the activity of ROI.
Acknowledgments
We thank Brenda Byrne and Lynne Shetron-Rama for technical assistance
with the culture of macrophages.
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