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Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan
Correspondence: Joel A. Swanson, Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620. E-mail: jswan{at}umich.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) and
lipopolysaccharide (LPS) leads to increased intracellular resistance to
microbes and increased major histocompatibility complex class
II-restricted antigen presentation, processes that both use the
vacuolar compartment. Despite the requirement of the macrophage
vacuolar compartment for microbicidal activities and antigen
processing, the rates of endocytosis and membrane trafficking in
activated macrophages are not clearly defined. In this study, vacuolar
compartment dynamics were analyzed in murine bone marrow-derived
macrophages activated with LPS and/or IFN-, conditions that
increased macrophage nitric oxide production and resistance to
infection by Listeria monocytogenes. Relative to
nonactivated cells, activated macrophages showed diminished rates of
fluid-phase pinocytosis and phagocytosis and delayed progression of
macropinosomes and phagosomes to late endosomes and lysosomes. In
contrast to the slowing of membrane trafficking, rates of macropinosome
acidification were similar between activated and nonactivated cells.
One consequence of this slowed membrane trafficking in activated
macrophages was a prolonged exposure of incoming molecules to an acidic
nonlysosomal compartment, a condition which may facilitate microbicidal
chemistries or antigen processing.
Key Words: macrophages • activation • Listeria monocytogenes • lipopolysaccharide • interferon- • lysosome
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Activating macrophages increases their destructive potential.
Macrophages obtained from mice previously exposed to sublethal doses of
Listeria monocytogenes can kill bacteria more effectively
than macrophages from unexposed mice [5
]. Macrophage
activation results primarily from exposure of macrophages to
interferon- (IFN-) and bacterial products, such as
lipopolysaccharide (LPS) or lipoteichoic acid [6
,
7
]. The enhanced ability to kill microorganisms is
largely a result of increased macrophage production of superoxide,
nitric oxide, and their derivatives [8
]. Although
lysosomes and lysosomal enzymes increase in number [9
],
it is not known if the lysosomes in activated macrophages are more
acidic or in some other way more cytotoxic. Resistance to L.
monocytogenes exhibited by IFN- treatment of peritoneal
macrophages is evidently because of blockage of escape from the
phagosome [10
]. Because escape typically occurs within
minutes of phagocytosis [11
], the mechanism of
resistance must reside in the earliest compartments of endocytosis.
Activation-related changes in vacuolar compartment functions may also
change the dynamics of those compartments. There is precedent for this
in the differentiation of dendritic cells. Undifferentiated dendritic
cells exhibit high rates of pinocytosis, and their differentiation into
antigen-presenting cells, stimulated by tumor necrosis factor 
,
interleukin-1ß, or LPS, is accompanied by a dramatic and irreversible
decrease in pinocytosis [12
]. It is not known if, upon
activation, macrophages undergo similar alterations in constitutive
endocytosis. Early studies indicated that activation increased rates of
pinocytosis. Edelson et al. [13
] reported high rates of
pinocytosis in thioglycollate-elicited mouse macrophages, and Knight et
al. [14
] observed an immediate stimulation of
pinocytosis in bone marrow-derived macrophages exposed to LPS. In
contrast, Montaner et al. [15
] found reduced rates of
fluid-phase pinocytosis in human monocyte-derived macrophages cultured
in IFN-. Thus, it is not clear whether activation increases or
decreases rates of membrane trafficking in macrophages.
The confused picture may result in part from differences between experimental systems for studying activation. It is widely acknowledged that there is no single differentiation state called activation; rather, different stimuli will lead to different pathways of differentiation, which are collectively referred to as activation [16 ]. Macrophages from different sources respond differently to activating stimuli, and a given kind of macrophage will exhibit different profiles of activation responses, depending on the nature of the activation stimulus. Our operational definition of an activated macrophage in this study was one with increased resistance to infection by L. monocytogenes, together with increased production of reactive nitrogen intermediates.
We hypothesize that the activated macrophage localizes its most potent
antimicrobial effects to phagosomes before they meet lysosomes, either
by creating novel compartments or by modifying the dynamics of existing
compartments. To define changes in membrane trafficking that
distinguish activated cells, we examined bone marrow-derived
macrophages activated with IFN- and LPS. After confirming that these
cells increased production of nitric oxide and resistance to L.
monocytogenes infection, we measured several features of organelle
trafficking. We provide evidence for a slowing of endocytosis and
membrane trafficking that allows a brief persistence of endocytosed
molecules in acidic nonlysosomal compartments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Genzyme, Cambridge, MA) and 10–1,000 ng/ml LPS (List Biological,
Campbell, CA) added to cells for 18–24 h. Washed sheep erythrocytes
(sRBC, Reproductive Sciences Program, University of Michigan, Ann
Arbor, MI) were opsonized for 45 min at 37°C using rabbit anti-sheep
erythrocyte IgG (ICN Biomedical, Aurora, OH), then were washed with
phosphate-buffered saline (PBS).
Measurement of nitrite in macrophages
Nitrite production by macrophages was measured using the Griess
assay, performed as described by Granger et al. [18
].
Briefly, after 24 h incubation of 1.0 x 105
cells in 0.75 ml DME-10F, plus or minus treatments, 0.7 ml cell
supernatant was removed and incubated with 0.7 ml Griess reagent
(0.05% napthylethylenediamine-HCl + 0.5% sulfanilamide in 1.25%
H3PO4) for 5 min. Absorbance was measured at
546 nm in a LKB Ultrospec II (Cambridge, England). Standard curves were
prepared using NaNO2 in DME-10F.
Measurement of Listeria monocytogenes growth in
macrophages
The ability of macrophages to kill L. monocytogenes
was assessed using the method of Jones and Portnoy [19
].
After overnight growth at room temperature in brain-heart infusion
broth (BHI; Difco, Detroit, MI), L. monocytogenes strain
10403S was diluted and further grown for 
1 h to 0.500
O.D600nm. After overnight treatment of 2.0 x
105 macrophages per well in 24-well plates, cells were
washed 3x in Ringers buffer (RB; 155 mM NaCl, 5mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 2 mM
NaH2PO4, 10 mM Hepes, and 10 mM glucose, pH
7.2), then incubated for 1 h in DME-10F without antibiotics. After
incubation, cells were incubated 30 min in 0.5 ml antibiotic-free
DME-10F plus bacteria. Infections were designed such that 5% of the
macrophages were infected with L. monocytogenes at the
earliest measured time point; this required that twice as many bacteria
be added to the activated macrophages (3 x 105
bacteria for control cells and 6 x 105 bacteria for
IFN- and LPS-treated cells; multiplicity of infection of 1.5 and 3,
respectively). After infection, cells were washed 4x in warm RB, then
incubated 0–5.5 h in DME-10F with 50 µg/ml gentamicin. Cells were
then washed 4x in warm RB and lysed in sterile deionized water.
Bacteria were then diluted and plated onto bovine heart infusion agar
for determination of colony-forming units.
To determine the relationship between inducible nitric oxide synthase (iNOS) expression, F-actin, and L. monocytogenes, macrophages on 13-mm coverslips were incubated overnight, with or without treatments. Cells were washed 3x in RB, then incubated in DME-10F without antibiotics for 1 h and infected as described above. They were then fixed 20 min with cytoskeletal fixative (40 mM HEPES, 10 mM EGTA, 0.5 mM EDTA, 5 mM MgSO4, 33 mM potassium acetate, 0.02% sodium azide, 5% polyethylene glycol 400, 4% paraformaldehyde). Cells were then washed 3x with PBS and permeabilized with 0.3% Triton X-100 in PBS for 5 min. Permeabilized cells were washed 3x for 5 min each, then incubated with mouse anti-iNOS (250 ng/ml) (Transduction, Lexington, KY) in PBS + 2% goat-serum (PBS-GS) overnight at 4°C. Cells were washed 3x for 5 min each with PBS-GS, then incubated with FITC anti-mouse IgG (Vector, Burlingame, CA), Texas Red-phalloidin (3.3 U/ml) (Molecular Probes, Eugene, OR), and DAPI (2 ng/ml) (Molecular Probes) for 2 h at 37°C. Cells were washed 3x for 5 min each with PBS-GS. Coverslips were mounted in glycerol and scored for the colocalization of DAPI-labeled L. monocytogenes with F-actin in iNOS-positive or -negative cells. Digital micrographs were taken using a Spot II cooled charge-coupled device camera (Diagnostic, Madison Heights, MI) on a Zeiss Axioplan II (Carl Zeiss, Thornwood, NY).
Pinocytosis in macrophages
Cells were plated at 2–3 x 105 cells per well
in 24-well plates and incubated overnight in experimental treatments.
After washing 3x with warm RB for 15 min, they were incubated with 0.5
mg/ml lucifer yellow CH (Molecular Probes) in warm RB for various
times, washed 2x for 5 min each in successive 2 x 1 L beakers of
cold PBS + 0.1% BSA, 1 L beaker of cold PBS, then lysed in 0.5 ml
of lysis buffer (0.1% Triton X-100, 50 mM Tris, pH 8.5). Fluorescence
of 0.4 ml lysate with 0.75 ml lysis buffer was measured in a
spectrofluorometer using excitation 430 nm and emission 540 nm.
Concentrations of lucifer yellow were determined using defined
concentrations of lucifer yellow in lysis buffer. Protein per well was
measured using the biscinchoninic acid protein assay (Pierce, Rockford,
IL).
Measurement of macropinosome and phagosome progression
To measure the rate at which macropinosomes and phagosomes
colocalize with LAMP-1, macrophages on 13-mm coverslips treated
overnight were washed 3x with RB, then were incubated for 2 min with 1
mg/ml fixable fluorescein dextran, Mr 10,000 (FDx10f;
Molecular Probes) plus 3,000 U/ml recombinant
macrophage-colony-stimulating factor (rM-CSF; Genetics Institute,
Cambridge, MA) or 3 min with 1 mg/ml FDx10f plus 1.0 x
107 opsonized sRBC. After washing 3x with RB, cells were
incubated for various times in RB. Cells were fixed with a modified
paraformaldehyde-lysine-periodate fixative ([20
], G-PLP:
20 mM MES, 70 mM NaCl, 5 mM KCl, 70 mM Lysine-HCl, 5 mM
MgCl2, 2mM EGTA, 10 mM NaIO4, 4.5% sucrose,
3.7% paraformaldehyde, pH 7.5) for 45 min at 37°C, then washed 3x
for 5 min with 20 mM Tris-HCl, 150 mM NaCl, 4.5% sucrose, pH 7.2,
supplemented with 2% goat serum (TBS-GS). Immunofluorescence was
performed by incubating cells with rat anti-LAMP-1 antibody
(Developmental Studies Hybridoma Bank, Iowa City, IA) for 1 h at
37°C. Cells were washed 3x for 5 min with TBS-GS, then incubated
with Texas Red-labeled anti-rat IgG (Vector) for 1 h at 37°C.
Cells were prepared for microscopy as described above. FDx10f-labeled
macropinosomes and phagosomes were scored positive when they were
completely labeled by LAMP-1.
To determine the rate of macropinosome fusion with lysosomes, macrophages on 13-mm coverslips were exposed to treatments overnight, then were incubated 30 min in DME-10F containing 0.5 mg/ml Texas Red Dextran, Mr 10,000 (TRDx10; Molecular Probes) to label endosomes. Cells were then incubated for 1 h in DME-10F to redistribute TRDx10 into lysosomes. Cells were washed 3x with RB and pulsed for 2 min with 1 mg/ml FDx10 and 3,000 U/ml M-CSF. Cells were rapidly washed 3x and incubated in RB for various times before fixation with G-PLP fixative for 45 min at 37°C and preparation for microscopy, as described above. FDx10-labeled macropinosomes were located and scored for complete colocalization with TRDx10.
To assess the rate of macropinosome formation, macrophages were incubated with FDx10 (1 mg/ml) plus M-CSF (3,000 U/ml) in RB for various times. Cells were quickly washed then fixed with G-PLP fixative for 45 min at 37°C, rinsed 3x for 5 min in TBS-GS, prepared for microscopy, then scored for the presence of FDx10-labeled macropinosomes.
Measurement of macropinosome pH
Macropinosome pH was measured by quantitative fluorescence
microscopy of FDx3-labeled organelles. To measure the pH in
macropinosomes, macrophages on coverslips were washed 3x and mounted
in a temperature-controlled stage at 37°C. Cells were pulsed with
0.75 mg/ml fluorescein dextran, Mr 3,000 (FDx3; Molecular
Probes) and 3,000 U/ml M-CSF in warm RB for 1.5 min, then washed
several times over 2.5 min in warm RB. Time-lapse images were acquired
by phase-contrast and fluorescence microscopy, with excitation of 485
and 440 nm and emission of 520 nm on a Zeiss IM-35 inverted microscope
(Carl Zeiss, Thornwood, NY). Images were analyzed with Metamorph image
analysis software (Universal, West Chester, PA). The 485-nm image was
thresholded to select the vacuole of interest, then a mask was created
to select the identical area in the 440-nm image. The pH of the vacuole
was calculated using a 485:440 nm ratio of the average vacuole
intensity calibrated to vacuoles incubated in 10 µM nigericin
(Molecular Probes) in isotonic potassium buffers of known pH (130 mM
KCl, 1 mM MgCl2, 15 mM Hepes, 15 mM MES, 0.02% sodium
azide) [11
].
Statistical analyses
Student’s two-tailed t-test was applied to data from
quantitative measurements, comparing data from nonactivated macrophages
to that from LPS-, IFN--, or LPS plus IFN--treated macrophages.
Figures show mean and standard error of the mean from all experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were activated by several measures. Production of nitrite
increased after treatment with either stimulant and increased
dramatically after exposure to both IFN- and LPS (Fig. 1A
). The increases in nitrite reflected nitric oxide production in
that its accumulation could be inhibited by 1 mM L-NMMA, a competitive
inhibitor of iNOS [21
]. Increases in cellular iNOS were
indicated by westerns of macrophage lysates and by immunofluorescence
localization of iNOS (data not shown). Independent measures of nitric
oxide production, using fluorescent sensors of nitric oxide, also
indicated increased nitric oxide production by activated macrophages
[22
]. Superoxide production after stimulation with
phorbol myristate acetate was also increased in the activated
macrophages (data not shown).
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plus LPS inhibited intracellular
growth of L. monocytogenes. The number of bacteria per
macrophage increased significantly more in nonactivated than activated
macrophages (Fig. 1B)
. The inhibition appeared to occur by prevention
of bacterial escape from phagosomes, because the percentage of bacteria
with associated F-actin, a measure of movement into the cytosolic space
[19
], was considerably reduced in the activated
macrophages (Fig. 1C)
.
Morphological changes accompanying activation
Macrophages activated with LPS and IFN- were a mixture of
well-spread and rounded cells, all of which were generally larger than
nonactivated macrophages. Time-lapse video microscopy revealed active
ruffling and the occasional formation of phase-bright macropinosomes
(data not shown). Vacuolar compartments labeled by pinocytosis of
fluorescein dextran appeared essentially similar to those of
nonactivated macrophages, consisting of small vesicles and tubular
lysosomes that extended radially from the perinuclear region
(Fig. 2
). A notable difference between the two kinds of macrophage was the
abundance in activated macrophages of phase-dense spherical granules
(Fig. 2)
, which were either absent in nonactivated macrophages or, when
present, were smaller than those of activated macrophages. The granules
were also abundant after exposure with LPS only and, to a lesser
extent, after exposure to IFN-. They appeared to be lipid granules
because they could not be labeled with any of a variety of endocytic
tracers and they were readily labeled with Nile Red, a stain for
neutral lipids (data not shown).
|
, LPS, or both (Fig. 3A
). The decreased accumulation apparently resulted from decreases
in the rate and extent of pinosome formation, as measurements of efflux
from cells preloaded with lucifer yellow showed no significant
differences between activated and nonactivated macrophages (data not
shown). Previous work determined that lucifer yellow and FDx10 are both
internalized by fluid-phase pinocytosis in macrophages
[4
, 23
], so their rates of accumulation and
efflux can be taken as indicators of fluid solute flux independent of
adsorptive or receptor-mediated components. Lucifer yellow was used to
measure pinocytosis by populations of cells, because methods for
measuring it in lysates are more sensitive than those for
fluorescein-dextrans.
|
2 fluorophores must
sometimes be compared in single cells, FDx10 is a preferable label for
pinosomes. To compare rates of pinosome formation, macrophages were
pulsed with FDx10 plus M-CSF for various intervals, then the fraction
of cells containing labeled macropinosomes was determined (Fig. 3B)
.
Activated macrophages formed macropinosomes more slowly, and a small
percentage of the cells did not produce macropinosomes at all. Figure 3B
indicates that nearly all activated macrophages made pinosomes,
which is consistent with the interpretation that all cells have
decreased rates of pinocytosis. The relative effects of M-CSF on
pinocytosis of activated and nonactivated macrophages were not
measured.
Delayed fusion with lysosomes
Activated macrophages also showed delayed progression of
macropinosomes and phagosomes to the lysosomal compartment. Rates of
progression were determined by measuring, at various times after their
formation, the fraction of pulse-labeled macropinosomes formed in the
presence of M-CSF or phagosomes that contained markers of late
endosomes or lysosomes. Lysosome-associated membrane protein-1 (LAMP-1)
served as a marker of late endosomes and lysosomes, and TRDx10, loaded
by pinocytosis and a 60-min chase, served as a marker for lysosomes.
Consequently, labeling for LAMP-1 allows measurement of pinosome
progression to late endosomes, and pulse-chase-labeling with TRDx10
shows progression to later compartments [24
]. Examples
of fused and unfused macropinosomes and phagosomes are shown in
Figure 4
. Immunofluorescence labeling for LAMP-1 appeared very similar to
the images of TRDx10-loaded late endosomes and lysosomes (data not
shown).
|
and LPS, the chase time required to label half of the
macropinosomes was 6 min longer than that required to label half the
pinosomes of nonactivated cells (Fig. 5A
B
). Macrophages activated with IFN- or LPS alone showed
intermediate progression rates, with LPS generally slowing things more
than IFN-. Macropinosome progression was difficult to assess after a
20 min chase, as nearly all macropinosomes had shrunken completely by
that time. In all macrophages, phagosomes containing IgG-opsonized
sheep erythrocytes acquired LAMP-1 nearly as quickly as macropinosomes
did; and like macropinosome progression, the phagosomes of activated
macrophages acquired LAMP-1 more slowly than the phagosomes of
nonactivated macrophages (Fig. 5C)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and LPS slow their endocytosis and membrane trafficking relative to
nonactivated cells. Activation increased some activities, such as
production of nitric oxide and resistance to intracellular growth of
L. monocytogenes, yet decreased organelle dynamics
generally. This slowing could be a counterproductive consequence of
producing toxic microbicidal compounds, or it could be a contributing
element of the cell’s microbicidal functions.
With exposure to LPS and IFN-, bone marrow-derived macrophages
acquire many features of activated primary macrophages
[6
]. Consistent with previous studies, exposure of bone
marrow-derived macrophages to IFN- and LPS increased production of
nitric oxide [25
]. Separate studies determined that this
protocol also increased superoxide production in response to phorbol
ester treatment (data not shown). These activated macrophages were also
more resistant to infection by L. monocytogenes, although
they were not as fully microbicidal as peritoneal macrophages treated
with IFN- [10
] (A. W. T., data not shown).
Different protocols for obtaining and activating macrophages result in
different levels of listericidal activity in macrophages
[26
]. The mechanisms underlying this heterogeneity in
responsiveness are not known but are likely to be related to the fact
that activation itself is not a singular differentiation event. In some
cases, IFN- alone is sufficient to increase listericidal activities
[10
], whereas in other cases, macrophages are only
listericidal after exposure to both IFN- and LPS
[27
]. Bone marrow-derived macrophages were selected for
these studies because exposure to IFN- and LPS increases measurably
their resistance to L. monocytogenes and their expression of
biochemical markers of activation, and because the flattened
morphologies of these cells render them optimal for quantitative
microscopic study.
The morphologies of activated macrophages were essentially as described previously, with one notable difference in the properties of the abundant phase-dense granules. Earlier studies characterized the phase-dense granules in LPS-activated macrophages as lysosomes [9 ]. However, the phase-dense granules seen here were apparently not part of the endocytic compartment, at least as defined by labeling with fluorescent tracers. Our preliminary studies indicate that they are lipid granules (J. A. S., data not shown).
The slowing of membrane trafficking observed in activated macrophages was measurable at several levels. Pinosomes formed more slowly, as did phagosomes (data not shown). Rates of fusion between pinosomes and lysosomes, and between phagosomes and lysosomes, were also reduced. These changes were not a result of decreased viability, as >95% of the activated macrophages continued to exclude propidium iodide (data not shown). The lower rates of phagosome-lysosome fusion contrast with the results of Kielian and Cohn [28 ], who observed increased rates of phagosome-lysosome fusion in macrophages elicited by peritoneal injection of Trypanosoma cruzi, Toxoplasma gondii, or proteose peptone. The different results may be a result of differences in methods of activation or of differences in the assays for phagosome-lysosome fusion.
The conclusion of this study, that endocytosis is slowed in activated
macrophages, is not fundamentally at odds with earlier studies that
reported increased pinocytosis in activated macrophages. Methodological
differences underlie the different conclusions. An earlier report of
increased pinocytosis in thioglycollate-elicited macrophages was
evidently not describing fluid-phase pinocytosis exclusive of
receptor-mediated endocytosis [13
]. That study measured
pinocytosis using HRP, a probe which was later shown to enter
macrophages by both fluid-phase pinocytosis and mannose
receptor-mediated endocytosis [29
] and to stimulate
fluid-phase pinocytosis itself [30
]. Increased
mannose-receptor-mediated endocytosis in thioglycollate-elicited
macrophages could explain the measured increases of HRP accumulation.
In another study, in which LPS increased rates of pinocytosis in bone
marrow-derived macrophages [14
], pinocytosis was
measured immediately after LPS addition, rather than much later, and
the data therefore would not reflect pinocytosis in activated
macrophages. The reduced rates of trafficking described here are
consistent with recent observations of Montaner et al.
[15
], in which prolonged exposure to IFN- led to a
general reduction in rates of pinocytosis in human monocytes. After
correcting for mannose-receptor-mediated endocytosis, they showed that
pinocytic accumulation of HRP by IFN--activated monocytes was
reduced relative to nonactivated control monocytes.
Pinocytic influx was slowed without attendant decreases in fluid-phase recycling. Various effects of cell stimulation on rates of influx and efflux have been noted previously [14 ]. It has been proposed that such changes can occur without net redistribution of cell membranes by changing the size ratios (i.e., average diameters) of influx and efflux vesicles [4 ].
The activated macrophages exhibited a decrease in membrane trafficking without a decrease in rates of vesicle acidification. This indicates that the H+-ATPase, that acidifies the vacuolar compartment, localizes to early endosomes in both activated and nonactivated macrophages.
The aggregate effect of activation with IFN- and LPS was a prolonged
residence of particles and macromolecules in acidic nonlysosomal
compartments (Fig. 7)
. Macropinosomes acidified rapidly but acquired
LAMP-1 and lysosomal content markers slowly. This alteration in the
dynamics of the compartments created a novel microenvironment without
creating a new organelle. This compartment may serve novel functions
peculiar to the activated macrophage. An acidic compartment deficient
in acid hydrolases could facilitate peptide loading onto MHC class II
molecules, or it could optimize antimicrobial chemistries involving
reactive oxygen or nitrogen species.
How important is a 6-min delay in endocytic vesicle progression to the
microbicidal activities of macrophages? This will most likely depend on
which microbe is considered. For the slow-growing intracellular
parasite Mycobacterium tuberculosis, changes in rates of
membrane trafficking may be of less consequence than biochemical
changes that would impair intracellular growth. Phagosomes containing
M. tuberculosis do not fuse with lysosomes in nonactivated
macrophages, but activation of macrophages with IFN- and LPS
increases delivery of M. tuberculosis-containing phagosomes
into the lysosomal compartment [31
]. This indicates that
activation causes a significant change in the dynamics of M.
tuberculosis-containing phagosomes specifically. For L.
monocytogenes, however, which escapes from phagosomes of
nonactivated macrophages shortly after phagocytosis
[11
], changes in early rates of phagosome progression to
lysosomes could affect the ability of bacteria to escape into
cytoplasm. Activation inhibits the ability of L.
monocytogenes to escape from the phagosome [10
].
Slowed phagosome maturation in activated macrophages of the magnitude
reported here could alter the nature of the enclosing membrane so as to
limit the ability of L. monocytogenes hemolysin to perforate
that membrane.
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ACKNOWLEDGEMENTS |
|---|
Received January 23, 2000; revised March 27, 2000; accepted April 10, 2000.
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and other signals regulate macrophage development Immunol. Rev. 97,5-27[Medline]
-Interferon limits access of Listeria monocytogenes to the macrophage cytoplasm J. Exp. Med. 170,2141-2146 or IL-10 J. Immunol. 162,4606-4613-induced macrophage activation Immunology 76,553-559[Medline]
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J. Zerrahn, U. E. Schaible, V. Brinkmann, U. Guhlich, and S. H. E. Kaufmann The IFN-Inducible Golgi- and Endoplasmic Reticulum- Associated 47-kDa GTPase IIGP Is Transiently Expressed During Listeriosis J. Immunol., April 1, 2002; 168(7): 3428 - 3436. [Abstract] [Full Text] [PDF]
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K. A. Christensen, J. T. Myers, and J. A. Swanson pH-dependent regulation of lysosomal calcium in macrophages J. Cell Sci., January 2, 2002; 115(3): 599 - 607. [Abstract] [Full Text] [PDF]
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 S. Sturgill-Koszycki and M. S. Swanson Legionella pneumophila Replication Vacuoles Mature into Acidic, Endocytic Organelles J. Exp. Med., October 30, 2000; 192(9): 1261 - 1272. [Abstract] [Full Text] [PDF]
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