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Inhibitory Effect of Toll-Like Receptor 4 on
Fusion between Phagosomes and
Endosomes/Lysosomes in Macrophages
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of June 18, 2017.
Akiko Shiratsuchi, Ikuko Watanabe, Osamu Takeuchi,
Shizuo Akira and Yoshinobu Nakanishi
J Immunol 2004; 172:2039-2047; ;
doi: 10.4049/jimmunol.172.4.2039
http://www.jimmunol.org/content/172/4/2039
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References
The Journal of Immunology
Inhibitory Effect of Toll-Like Receptor 4 on Fusion between
Phagosomes and Endosomes/Lysosomes in Macrophages1
Akiko Shiratsuchi,*† Ikuko Watanabe,† Osamu Takeuchi,‡ Shizuo Akira,‡ and
Yoshinobu Nakanishi2*†
C
ells undergoing apoptosis are selectively and rapidly
eliminated from organisms by phagocytosis (reviewed in
Refs. 1 and 2). This process contributes not only to the
removal of unnecessary or spent cells, but also to maintaining tissue homeostasis (reviewed in Refs. 3 and 4). Examples of
processes involved in the latter function include the following:
possible autoantigens (reviewed in Refs. 5 and 6) and proinflammatory substances are eliminated before they leak out from dying
cells; production of viral progeny is inhibited when virus-infected
cells are phagocytosed by macrophages (7); anti-inflammatory cytokines are secreted from macrophages engulfing apoptotic cells
(reviewed in Ref. 8); dendritic cells present Ag that exist in engulfed apoptotic cells (reviewed in Refs. 9 and 10); and testicular
Sertoli cells control spermatogenesis by eliminating apoptotic
spermatogenic cells (11). Phagocytic clearance of apoptotic cells
consists of four distinct steps: accumulation of phagocytes at the
site where apoptotic cells are located, recognition of apoptotic cells
by phagocytes, engulfment of apoptotic cells, and processing of
engulfed apoptotic cells in phagocytes. Among these, only the recognition step has been intensely studied, and candidate molecules
*Graduate School of Medical Science and †Graduate School of Natural Science and
Technology, Kanazawa University, Kanazawa, Ishikawa, Japan; and ‡Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
Received for publication May 14, 2003. Accepted for publication November
25, 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
This work was supported by a Grant-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science, a grant from the Hayashi Memorial Foundation
for Female Natural Scientists, a grant from the Nissan Science Foundation, and a
grant from the Hokkoku Foundation.
2
Address correspondence and reprint requests to Dr. Yoshinobu Nakanishi, Graduate
School of Medical Science, Kanazawa University, Takara-machi, Kanazawa, Ishikawa 920-0934, Japan. E-mail address: [email protected]
Copyright © 2004 by The American Association of Immunologists, Inc.
responsible for phagocyte binding to apoptotic cells have been
proposed (reviewed in Refs. 4, 12, and 13).
Immunity is central to the self-defense mechanism, which can
be divided into the innate and adaptive immune responses. Innate
immunity is defined as a mechanism by which self and non-self are
discriminated without involvement of proteins encoded by genes
that are rearranged during development (reviewed in Refs. 14 and
15). A major role for innate immunity is front-line protection of
organisms from invasion by pathogenic microbes: leukocytes directly eliminate invading microbes by recognizing molecules
(which are called pathogen-associated molecular patterns) present
on the surface of the invaders and activate the adaptive immune
response (reviewed in Refs. 14 and 15). Toll-like receptors (TLR)3
are included among the receptors responsible for leukocyte recognition of microbes (reviewed in Refs. 16 –18).
Recognition of apoptotic cells by phagocytes does not involve,
as known to date, either Ig or TCR, and therefore phagocytic clearance of apoptotic cells can be considered as an innate immune
response (reviewed in Ref. 19). However, no substantial link between the two phenomena of innate immunity and apoptotic cell
removal has been demonstrated. Macrophages respond to Gramnegative bacteria by recognizing the endotoxin LPS present on the
surface of the bacteria, and this recognition requires the presence
of both TLR4 and CD14 on the surface of macrophages (reviewed
in Ref. 20). It has also been suggested that CD14 plays a role in the
recognition of apoptotic cells by macrophages (reviewed in Refs.
4, 12 and 13). We therefore anticipated that TLR4 might participate in the phagocytosis of apoptotic cells by macrophages. To test
this possibility, we examined the phagocytosis of apoptotic cells
by macrophages prepared from tlr4-disrupted mice.
3
Abbreviations used in this paper: TLR, Toll-like receptor; DHMEQ, dehydroxymethylepoxyquinomicin; PC, phosphatidylcholine; PI3K, phosphatidylinositol 3-kinase; PS, phosphatidylserine.
0022-1767/04/$02.00
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Toll-like receptor 4 (TLR4) of macrophages recognizes LPS of Gram-negative bacteria in cooperation with CD14, which is also
involved in the recognition of apoptotic cells. In this study we asked whether TLR4 plays a role in the phagocytic clearance of
apoptotic cells by macrophages. Macrophages were prepared from peritoneal fluid of thioglycolate-treated mice carrying either
a wild-type or a disrupted TLR4-encoding gene and were examined for their ability to phagocytose apoptotic mouse thymocytes,
apoptotic Jurkat T cells, Ig-opsonized mouse thymocytes, Ig-opsonized zymosan particles, and latex beads. Both populations of
macrophages equally expressed CD14 on their surfaces and showed almost equal activities of binding to and engulfing all these
targets. However, apoptotic thymocytes, apoptotic Jurkat cells, and opsonized thymocytes disappeared more rapidly in TLR4deficient macrophages than in wild-type macrophages, and the fusion between endosomes/lysosomes and phagosomes containing
any target cells or particles was accelerated in mutant macrophages. Activation of the transcription factor NF-␬B appeared not
to occur in wild-type macrophages after engulfment, and the rate of apoptotic cell degradation in wild-type macrophages remained
the same regardless of the activation of NF-␬B. Finally, immunohistochemical analyses showed that ectopically expressed TLR4
was associated with phagosomes in a macrophage-derived cell line. All these results collectively indicate that TLR4 negatively
regulates the degradation of engulfed cells in macrophages via a pathway independent of NF-␬B. The Journal of Immunology,
2004, 172: 2039 –2047.
2040
INHIBITION OF PHAGOSOME-LYSOSOME FUSION BY TLR4
Materials and Methods
Analysis of endosomes/lysosomes
Mice and cells
To locate endosomes/lysosomes immunohistochemically, macrophages
were successively treated with paraformaldehyde and methanol, and then
incubated with Ab recognizing either the endosomal/lysosomal membrane
protein LGP85 (26) or the lysosomal enzyme cathepsin D (27). They were
then washed, treated with FITC-labeled anti-rabbit IgG Ab (Vector Laboratories), and examined by confocal laser scanning microscopy (LSM510
system; Carl Zeiss, Jena, Germany). Endosomes/lysosomes of macrophages were also detected histochemically under a fluorescence microscope after treatment with cell-permeable fluorochromes, Acridine Orange
(Sigma-Aldrich), and Lysotracker Red (Molecular Probes), which accumulate in acidic granules, and Texas Red-labeled dextran (m.w., 70,000;
lysine fixable; Molecular Probes), which is actively transported to these
organelles. For determination of the activity of the lysosomal enzyme ␣-Nacetylglucosaminidase, whole-cell lysates of peritoneal macrophages were
incubated in 0.1 M sodium acetate buffer (pH 5.5) containing 4-methylumbelliferyl N-acetyl-␣-D-glucosaminide (50 nM; Sigma-Aldrich) at 37°C
for 30 min. The amount of cleaved substrate was determined using a fluorometer with excitation and emission at 365 and 450 nm, respectively.
Phagocytosis assay
Phagocytosis of mouse thymocytes or Jurkat cells by macrophages was
examined essentially as described previously (22, 25). Briefly, target cells
were mixed with macrophages (at a ratio of 10 target cells to one macrophage) and were incubated at 37°C for various lengths of time. The mixture
was first agitated by pipetting and then treated with trypsin to remove target
cells that were lightly attached to macrophages. The macrophages that
remained attached to coverslips were fixed, permeabilized, and stained
with hematoxylin as described previously (25). The numbers of macrophages containing engulfed target cells and of engulfed target cells present
in each macrophage were determined by examination under a light microscope, and expressed relative to total macrophages (in percentage; the
phagocytic index) and per 100 macrophages, respectively. To determine
the efficiency of macrophage binding to target cells, macrophages were
incubated with thymocytes or Jurkat cells as described above, fixed without
pipetting and trypsin treatment, and stained with hematoxylin. The number
of target cells bound to macrophages was then determined by microscopic
examination. For examination of the PS dependency of phagocytosis, liposomes consisting of either phosphatidylcholine (PC) alone or a mixture
of PC and PS (at a molar ratio of 7:3) were included in phagocytosis
reactions at various concentrations (22). The involvement of phosphatidylinositol 3-kinase (PI3K) was examined by preincubating macrophages with
10 nM wortmannin (Sigma-Aldrich) for 1 h before phagocytosis or binding
assay. To determine the extent of degradation of thymocytes engulfed in
macrophages, the phagocytosis assay was conducted in the presence of 0.1
mM chloroquine (Sigma-Aldrich), an inhibitor of the acidification of endosomes/lysosomes. To examine the phagocytic activity of macrophages
against mouse IgG-opsonized and FITC-labeled zymosan particles (zymosan A; Sigma-Aldrich) or FITC-labeled latex beads (Polybead Microparticles; Polysciences, Warrington, PA), macrophages were incubated with
target particles, washed with PBS, and fixed. The extent of incorporation of
targets was then determined by examination under a fluorescence microscope (BX50; Olympus, Tokyo, Japan). Scores for all the above measurements are shown as the mean and SD of a typical example from at least
three independent experiments.
Analysis of CD14 expression
The expression of CD14 on the surface of macrophages was examined by
immunofluorescence and flow cytometry using anti-mouse CD14 mAb
(clone rmC5-3; Research Diagnostics, Flanders, NJ). Unfixed macrophages
were first treated with anti-CD14 Ab, then successively with biotin-labeled
anti-rat IgG Ab (Vector Laboratories, Burlingame, CA) and Alexa 488conjugated streptavidin (Molecular Probes, Eugene, OR). They were examined by fluorescence microscopy or flow cytometry (EPICS-XL;
Coulter, Hialeah, FL). Macrophages treated with IgG prepared from normal rat serum were similarly analyzed as a negative control.
Phagosome-endosome/lysosome fusion assay
Macrophages whose endosomes/lysosomes had been loaded with either
Acridine Orange (assay A) or Lysotracker Red (assay B) were subjected to
a phagocytosis assay with various target cells or particles. In assay A,
Acridine Orange-stained macrophages were mixed with unlabeled targets
and examined by fluorescence microscopy. Endosomes/lysosomes not
fused with phagosomes were stained orange with dimerized acridine orange, whereas those after fusion turned green because the fluorochrome
was diluted and converted to greenish monomers (28). The number of
macrophages containing fused granules was determined and expressed (in
percentage) relative to total macrophages. In assay B, macrophages loaded
with Lysotracker Red were incubated with apoptotic thymocytes, apoptotic
Jurkat cells, or IgG-opsonized thymocytes that had been labeled with another fluorescent dye, PKH2 (Zynaxis, Malvern, PA), or with unlabeled
IgG-opsonized zymosan particles or latex beads. Unfused phagosomes that
contained PKH2-labeled target cells were seen in green, whereas those
fused with endosomes/lysosomes were seen in yellow due to the coexistence of the two fluorochromes. In contrast, intracellular distribution of
unlabeled zymosan particles or latex beads in macrophages was determined
by fluorescence phase contrast microscopy, and engulfed target-containing
granules existing apart from and being colocalized with Lysotracker Redlabeled endosomes/lysosomes were considered as unfused and fused
phagosomes, respectively. The number of fused phagosomes was determined in this way and expressed (in percentage) relative to total phagosomes. Scores in either assay are shown as the mean and SD of a typical
example from at least two independent experiments.
Expression and detection of epitope-tagged TLR4
RAW264.7 cells were transfected with pEFBOS/mouse TLR4flaghis, an
expression vector for mouse TLR4 fused with FLAG and His tags (29), by
Lipofection using GenePORTER2 (Gene Therapy Systems, San Diego,
CA). The resulting cells were incubated with unlabeled latex beads (Ø ⫽
1.053 ␮m) or apoptotic thymocytes at 37°C for 0.5–2 h. The cells were
then successively reacted with anti-FLAG mAb M2 (Sigma-Aldrich),
biotin-labeled anti-mouse IgG, and Alexa 488- or Alexia 546-conjugated
streptavidin (Molecular Probes), followed by examination under a confocal
laser-scanning microscope.
Preparation of nuclear extracts and EMSA
Nuclear extracts were prepared from macrophages before and after phagocytosis of apoptotic mouse thymocytes according to the method described
by Schreiber et al. (30). As a positive control, nuclear extracts were prepared from macrophages that were incubated with LPS of Salmonella enteriditis (10 ␮g/ml) (Sigma-Aldrich) at 37°C for 1 h. A double-stranded
oligonucleotide with the sequence 5⬘-ATGTGAGGGGACTTTCCCAGGC,
which contains the binding sequence for NF-␬B in the mouse ␬ L chain enhancer, was labeled with 32P at the 5⬘ ends and used as a probe in an EMSA.
The extracts were first incubated with sonicated salmon testis DNA (3 mg/ml)
for 10 min on ice and then reacted with the 32P-labeled probe (10 nM) for 10
min on ice. When necessary, after the binding reaction the mixture was further
incubated in the presence of anti-NF-␬B p65 subunit Ab (sc-109; Santa Cruz
Biotechnology, Santa Cruz, CA; 0.2 mg/ml IgG) or anti-C/EBP-␤ Ab (sc-150;
Santa Cruz Biotechnology; 0.2 mg/ml IgG) for 1 h on ice. The samples were
loaded onto a 6% (w/v) polyacrylamide gel containing 5% (v/v) glycerol and
resolved by electrophoresis at 10 V/cm at 4°C in 25 mM Tris-borate (pH 8.3)
containing 0.5 mM EDTA. The gel was dried on DEAE paper (Advantec,
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Mice carrying a disrupted tlr4 in a C57BL/6 background, which neither
express TLR4 nor respond to LPS, were generated previously (21). Mice
with a normal tlr4 obtained by breeding in our laboratory or C57BL/6 mice
purchased from a commercial source were used as the wild-type control.
Macrophages were prepared from peritoneal fluids of thioglycolate-injected mice carrying either a wild-type or disrupted tlr4 and were cultivated
on coverslips until used as described previously (22, 23). Thymocytes prepared from ddY mice (female, 6 – 8 wk old) were cultured in RPMI 1640
containing 10% (v/v) FBS at 37°C with 5% (v/v) CO2 in air. They were
incubated with 10 ␮M dexamethasone (Sigma-Aldrich, St. Louis, MO) to
induce apoptosis. Jurkat cells, a line of human leukemia T cells, maintained
in the same medium as thymocytes were treated with doxorubicin (SigmaAldrich; 0.3 ␮g/ml) for 24 –30 h to induce apoptosis. Both dexamethasonetreated thymocytes and doxorubicin-treated Jurkat cells showed features
typical of apoptotic cells, including exposure of the membrane phospholipid phosphatidylserine (PS) on the cell surface. To opsonize thymocytes
with Ig, dexamethasone-treated thymocytes were incubated with antimouse Thy1 IgG mAb G7 (24) for 20 min, and binding of the Ab to
thymocytes was confirmed by fluorescence microscopy after treating the
cells with FITC-labeled anti-mouse IgG Ab. RAW264.7 cells, a mouse
macrophage-derived cell line, were maintained in DMEM containing 10%
(v/v) heat-inactivated FBS at 37°C with 5% (v/v) CO2 in air. All experiments involving animals were conducted with the approval of the committee on animal experimentation of Kanazawa University, Takara-machi
Campus.
The Journal of Immunology
Tokyo, Japan), and the signals were visualized using an image analyzer
(BAS1000; Fuji Photo Film, Minami-Ashigara, Japan).
Results
Rapid decrease in the level of phagocytosis of apoptotic cells by
TLR4-deficient macrophages
No change in the mode of phagocytosis by TLR4-deficient
macrophages
The loss of TLR4 could alter the expression of CD14 in macrophages. We thus immunochemically examined the cell surface expression of CD14 in wild-type and TLR4-deficient macrophages
using anti-mouse CD14 Ab. The results showed that there is no
FIGURE 1. Decrease in the level of
phagocytosis of apoptotic cells by TLR4deficient macrophages at late stages of incubation. Peritoneal macrophages with a
wild-type or disrupted tlr4 (TLR4⫺/⫺) were
used in the phagocytosis assay with mouse
thymocytes undergoing dexamethasone-induced apoptosis (A), Jurkat T cells undergoing doxorubicin-induced apoptosis (B),
IgG-opsonized mouse thymocytes (C),
IgG-opsonized zymosan particles (D), or latex beads (E). The extent of phagocytosis
was determined at the indicated times, and
the data are shown as the mean ⫾ SD (n ⫽
3– 6). Shown on the left in A are hematoxylin-stained, wild-type macrophages that
contain engulfed thymocytes at various
time points of the phagocytosis reaction,
and the arrowheads denote thymocytes that
became smaller in size and more lightly
stained with hematoxylin.
significant difference in the distribution (Fig. 2A) or the level of
expression (Fig. 2B) of cell surface CD14 between the two macrophage preparations. This indicates that CD14 is not responsible
for the observed change in phagocytosis by TLR4-deficient macrophages. We next compared the modes of phagocytosis of apoptotic cells by TLR4-deficient vs wild-type macrophages (Fig. 3).
Both macrophage preparations efficiently phagocytosed dexamethasone-treated thymocytes, but normal thymocytes were relatively
poor targets (Fig. 3A). The addition of liposomes containing PS,
but not those consisting of PC alone, severely inhibited the phagocytosis of apoptotic thymocytes by macrophages regardless of the
presence of TLR4 (Fig. 3B). Finally, the addition of the PI3K
inhibitor wortmannin inhibited the engulfment, but not the binding, of apoptotic thymocytes by macrophages, and there was no
significant difference in the extent of inhibition between the two
macrophage populations (Fig. 3C). These results indicate that
TLR4 deficiency does not alter the mode of macrophage phagocytosis of apoptotic thymocytes, i.e., the dependency on apoptosis
induction in thymocytes, the requirement for macrophage recognition of PS exposed on the surface of apoptotic thymocytes, and
the requirement for PI3K activity in macrophages. Furthermore,
apoptotic thymocytes bound to TLR4-deficient and wild-type macrophages with similar efficiencies (see Fig. 3C). These results indicate that TLR4 deficiency affects macrophage phagocytosis at a
step(s) other than recognition and engulfment.
Abrogation of the effect of TLR4 deficiency in the presence of
endosome/lysosome inhibitor
We then tested the possibility that engulfed thymocytes are processed differently in wild-type and TLR4-deficient macrophages.
For this purpose, phagocytosis reactions with apoptotic thymocytes were conducted in the presence of inhibitors of endosomal/
lysosomal acidification; this allowed us to monitor phagocytosis
reactions only up to the step of engulfment. Among three reagents
tested, ammonium chloride, chloroquine, and bafilomycin A1,
only chloroquine was not toxic to peritoneal macrophages (data
not shown). We therefore conducted phagocytosis reactions in the
presence or the absence of this drug and determined the extent of
phagocytosis (Fig. 4). In the absence of chloroquine, the phagocytic index decreased during the late stage of the time course, and
the rate of this decrease was higher in reactions with TLR4-deficient macrophages (Fig. 4A), as also described above. In contrast,
inclusion of the inhibitor caused the phagocytic index to persist at
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The effects of the absence of TLR4 on macrophage function of
phagocytosing apoptotic cells were examined. To do this, macrophages were prepared from peritoneal fluids of thioglycolatetreated mice carrying either a wild-type or a disrupted tlr4 and were
subjected to a phagocytosis assay with mouse thymocytes undergoing
dexamethasone-induced apoptosis (Fig. 1A). When the time course
was determined with wild-type macrophages, both the relative number of macrophages engulfing thymocytes, i.e., the phagocytic index,
and the number of engulfed targets in macrophages increased linearly
during the initial stage of incubation, reached a maximum level at
0.5 h, and gradually decreased thereafter. The decrease in the level of
phagocytosis at the late stage of incubation is most likely due to degradation of engulfed thymocytes in macrophages, because thymocytes
that were smaller in size and more lightly stained with hematoxylin
appeared after 2 h (left panels in Fig. 1A). The time course of phagocytosis reactions with TLR4-deficient macrophages was indistinguishable from that with wild-type macrophages until the extent of the
reaction reached a maximum. However, the level of phagocytosis, in
terms of both the ratio of target cell-engulfing macrophages and the
number of target cells engulfed by macrophages, by mutant macrophages was significantly lower than that by wild-type macrophages
beyond this maximum point. This was also the case, although less
striking, in the reactions with Jurkat T cells undergoing doxorubicininduced apoptosis (Fig. 1B) or IgG-opsonized mouse thymocytes
(Fig. 1C; similar time courses were obtained when the number of
engulfed target cells in macrophages was measured). Such effects of
TLR4 deficiency were, however, not observed in macrophages that
engulfed IgG-opsonized zymosan particles (Fig. 1D) or indigestible
latex beads (Fig. 1E). These results suggest that TLR4 deficiency
causes rapid degradation of engulfed cells in macrophages.
2041
2042
a level near the maximum even at late stages of incubation, and the
two macrophage preparations did not show a significant difference
in this regard (Fig. 4B). These results indicate that endosomes/
lysosomes need to be active for TLR4 deficiency to affect the macrophage phagocytosis of apoptotic thymocytes. It seems probable
that lysosomal degradation of engulfed thymocytes is accelerated
in macrophages that are deficient in TLR4 expression.
No change in the intracellular distribution or function of
endosomes/lysosomes in TLR4-deficient macrophages
We next asked whether TLR4 deficiency causes altered distribution and activity of endosomes/lysosomes in macrophages. The
intracellular distribution of endosomes/lysosomes was analyzed
histochemically using either Ab that recognize endosomal/lysosomal proteins or fluorochromes that accumulate in these organelles.
When macrophages were analyzed immunohistochemically with
anti-LGP85 or anti-cathepsin D Ab, they gave a staining pattern
typical of endosomes/lysosomes in macrophages (31, 32) (Fig.
5A). Endosomes/lysosomes were similarly detected by treatment
with the fluorochromes Acridine Orange and Lysotracker Red,
which accumulate in acidic compartments, including endosomes/
lysosomes (Fig. 5B), or with fluorescence-labeled dextran, which
is actively transported to these organelles (Fig. 5C). There appeared to be no difference in the distribution of signals in any
examinations between TLR4-deficient and wild-type macrophages. We next examined the function of lysosomes by determining the activity of ␣-N-acetylglucosaminidase, a representative
lysosomal enzyme, in whole-cell lysates of macrophages using a
fluorescence-labeled substrate. The level of the enzyme activity
was almost the same in lysates of the two macrophage populations
(Fig. 5D), indicating that lysosomes in TLR4-deficient macrophages are functionally normal. These results indicate that the loss
of TLR4 does not alter the morphological appearance or the function of endosomes/lysosomes in macrophages.
Enhancement of the phagosome-endosome/lysosome fusion in
TLR4-deficient macrophages
We then examined the effect of TLR4 deficiency on the fusion
between phagosomes and endosomes/lysosomes in macrophages.
For this purpose, wild-type macrophages that had been incubated
with endosome/lysosome-targeting fluorochromes were subjected
to phagocytosis reactions with apoptotic thymocytes and examined
for the occurrence of the fusion in two different assays. In one
assay (assay A), macrophages incubated with Acridine Orange
were mixed with unlabeled apoptotic thymocytes. In those macrophages, unfused endosomes/lysosomes were stained red, and
many phagosomes (indicated by an arrow in the top panels of Fig.
6A) and macrophage nuclei (indicated by an asterisk) were seen in
red and light green, respectively, probably due to the adsorption of
Acridine Orange. There were some granules stained in green (indicated by an arrowhead), and these were most likely phagosomes
fused with endosomes/lysosomes, in which Acridine Orange was
diluted and converted from red dimers to green monomers. The
number of macrophages containing green granules was determined
and expressed relative to total macrophages to indicate the extent
of the fusion. In the other assay (assay B), macrophages incubated
with Lysotracker Red were fed apoptotic thymocytes labeled with
the green fluorescent dye PKH2. These macrophages possessed red
endosomes/lysosomes and greenish particles, the latter of which
were considered to be phagosomes containing engulfed thymocytes (indicated by an arrow in the bottom panels of Fig. 6A).
Fusion of thymocyte-containing phagosomes with endosomes/lysosomes resulted in the colocalization of Lysotracker Red and
green PKH2, which caused the fused phagosomes to turn yellow
(indicated by an arrowhead). The number of yellow phagosomes
was determined and expressed relative to total phagosomes (green
plus yellow) to indicate the efficacy of the fusion. The extent of the
fusion was compared in this way between wild-type and TLR4dificient macrophages during incubation with various target cells,
except that in assay B the presence of fused phagosomes was determined by fluorescence/phase contrast microscopy in reactions using
unlabeled opsonized zymosan or latex beads as target particles. The
results of these two assays almost coincided with each other and
showed that the fusion between phagosomes and endosomes/
lysosomes takes place more efficiently in TLR4-deficient macrophages than in wild-type macrophages using any target cells or
particles (Fig. 6B). We therefore concluded that TLR4 acts to suppress the fusion between phagosomes and endosomes/lysosomes
regardless of the type of target cell. This explains why degradation of
engulfed cells is accelerated in TLR4-deficient macrophages. Rapid
disappearance of zymosan particles in TLR4-deficient macrophages
was not obvious, probably because they were only slowly degraded in
endosomes/lysosomes.
Independence of apoptotic cell degradation in macrophages
from NF-␬B
Binding of LPS, an endotoxin produced in Gram-negative bacteria,
to CD14/TLR4 of macrophages leads to translocation of the transcription factor NF-␬B from the cytoplasm to the nucleus and thus
to activation of NF-␬B-mediated gene transcription (reviewed in
Refs. 33–35). We therefore examined whether nuclear translocation of NF-␬B occurs in wild-type macrophages after engulfment
of apoptotic cells. For this purpose, nuclear extracts were prepared
from macrophages either treated or not treated with apoptotic cells
and examined for the presence of NF-␬B by EMSA (Fig. 7A).
When nuclear extracts of LPS-treated macrophages were analyzed
as a positive control, a distinct signal whose electrophoretic mobility was slower than that of the free probe was obtained. This
signal was shown to be a complex formed between the probe and
NF-␬B, because a decrease in the intensity of the signal and the
appearance of a supershift signal occurred in the presence of antiNF-␬B Ab, but not of unrelated Ab (C/EBP). The DNA-binding
activity of NF-␬B was present, although at a trace level, in nuclear
extracts of untreated macrophages, and its level did not significantly change after incubation with apoptotic mouse thymocytes.
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FIGURE 2. No difference in the distribution and level of cell surface
CD14 between wild-type and TLR4-deficient macrophages. A, Immunofluorescence. Wild-type and TLR4-deficient peritoneal macrophages were
subjected to immunofluorescence using anti-mouse CD14 Ab or control rat
IgG. Fluorescence and phase contrast views of the same microscopic fields
are shown. The scale bar represents 10 ␮m. B, Flow cytometry. Macrophages treated with either anti-CD14 or control Ab were analyzed by flow
cytometry. Vertical dotted lines indicate the position of peak fluorescence
in the analysis with control IgG.
INHIBITION OF PHAGOSOME-LYSOSOME FUSION BY TLR4
The Journal of Immunology
2043
We then examined whether NF-␬B activity is required for regulation of the degradation of engulfed cells by TLR4. Wild-type
macrophages were preincubated for 2 h with dehydroxymethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear translocation of
NF-␬B (36), and were subjected to a phagocytosis assay with apoptotic mouse thymocytes in the presence of DHMEQ. The inclusion of this drug completely abolished the accumulation of NF-␬B
in the nucleus of LPS-treated macrophages (data not shown). The
level of phagocytosis indexes, however, was not significantly different regardless of the presence of the inhibitor at any time point
examined (Fig. 7B). Furthermore, the treatment with LPS, which
induces NF-␬B activation, did not alter the phagocytic activity of
macrophages (Fig. 7C). The results were essentially the same
when the level of phagocytosis was determined by measuring the
number of thymocytes engulfed by macrophages (data not shown).
These results collectively indicate that NF-␬B is not involved in
TLR4-mediated regulation of the degradation of engulfed apoptotic cells in macrophages.
Association of TLR4 with phagosomes in macrophages
We finally determined the localization of TLR4 in macrophages
after engulfment. As Ab that can be used for immunohistochemical
detection of TLR4 in mouse cells was not available, we ectopically
expressed epitope-tagged TLR4 in RAW264.7 cells, a mouse macrophage-derived cell line, so that localization of the protein could
be determined using an Ab that recognizes the epitope. The
epitope-tagged TLR4 was present at the cell surface as well as in
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FIGURE 3. No difference in the mode of phagocytosis between wild-type and TLR4-deficient macrophages. Wild-type and TLR4-deficient peritoneal
macrophages were subjected to the phagocytosis assay with mouse thymocytes, and the modes of the phagocytosis reactions were compared. A, Dependency
on apoptosis in target cells. Macrophages were incubated for 30 min with thymocytes that had been treated (⫹) or not treated (⫺) with dexamethasone,
and the extent of phagocytosis was determined. The data are the mean ⫾ SD (n ⫽ 4). B, Macrophage recognition of PS exposed on the surface of target
cells. Phagocytosis reactions were conducted with dexamethasone-treated thymocytes for 1 h in the presence of PS-containing liposomes at various
concentrations, and the extent of phagocytosis was determined and is shown relative to that in reactions with no added liposomes. The values obtained for
reactions with wild-type macrophages in the presence of control liposomes consisting of PC alone (1 mM) are shown. The data are the mean ⫾ SD (n ⫽
4). C, Requirement for the activity of PI3K in macrophages. Macrophages that had been preincubated for 1 h in the presence (⫹) or the absence (⫺) of
wortmannin were further incubated for 1 h with dexamethasone-treated thymocytes. Microscopic views of bound and engulfed thymocytes (indicated by
arrowheads) are shown on the left. The scale bar represents 10 ␮m. The numbers of thymocytes attached to (binding) or engulfed by (engulfment)
macrophages were determined and are shown relative to the number in reactions with no added inhibitor. The numbers of bound thymocytes (per 100
macrophages) was 30 ⫾ 5 and 36 ⫾ 10 for wild-type and TLR4-deficient macrophages, respectively. The data are the mean ⫾ SD (n ⫽ 4).
2044
FIGURE 4. Abrogation of the effect of TLR4 deficiency in the absence
of endosome/lysosome function. Phagocytosis of apoptotic mouse thymocytes by wild-type or TLR4-deficient macrophages was conducted in the
absence (A) or the presence (B) of chloroquine, an inhibitor of endosome/
lysosome acidification, which was added 1 h before and continued to be
present during the reaction. The data are the mean ⫾ SD (n ⫽ 6).
Discussion
Phagocytic clearance of apoptotic cells may be considered a part of
innate immunity. We have provided here for the first time evidence
that a molecule that functions in the innate immune response
against invading microbes is also involved in the phagocytosis of
apoptotic cells by macrophages. As a pattern recognition receptor
in innate immunity, TLR4 recognizes Gram-negative bacteria by
binding to the endotoxin LPS in cooperation with CD14. As it has
also been suggested that CD14 is involved in the recognition of
apoptotic cells, we anticipated that TLR4 would play a role in
phagocytic clearance of apoptotic cells. Our expectation was that
the participation of TLR4, if it occurs at all, would be involved in
the recognition of apoptotic cells by macrophages. The results
showed, however, that TLR4 plays a role in macrophage phagocytosis of apoptotic cells, not in the recognition step but, rather, in
the step of lysosomal degradation of engulfed cells, by regulating
fusion between phagosomes and endosomes/lysosomes.
It is unclear at present how TLR4 regulates phagosome-endosome/lysosome fusion in macrophages. When TLR4 functions as a
pattern recognition receptor in innate immunity, it transduces signals leading to the activation of the transcription factor NF-␬B
(reviewed in Refs. 35 and 36). However, our results indicate that
the regulation of phagosome-endosome/lysosome fusion by TLR4
does not require the activity of NF-␬B. Taking into consideration the
fact that the lack of TLR4 did not affect the recognition and engulfment of target cells by macrophages, TLR4 is likely to function within
macrophages to regulate the degradation of engulfed cells. In fact, we
showed that ectopically expressed TLR4 was associated with
phagosomes containing either apoptotic thymocytes or latex beads
in a macrophage-derived cell line. Another TLR family member,
FIGURE 5. No change in the intracellular distribution and function of endosomes/lysosomes in TLR4-deficient macrophages. A, Immunohistochemical
detection of endosomes/lysosomes in macrophages. Macrophages were subjected to immunofluorescence with anti-LGP85 or anti-cathepsin D Ab. Phase
contrast and fluorescence views of the same microscopic fields are shown. The scale bars represent 10 ␮m. B, Detection of endosomes/lysosomes with acidic
granule-targeting fluorochromes. Macrophages were treated with Acridine Orange or Lysotracker Red and examined by fluorescence microscopy. Fluorescence microscopic views are shown. The scale bar represents 10 ␮m. C, Detection of endosomes/lysosomes with an endosome/lysosome-targeting dye.
Macrophages were treated with Texas Red-labeled dextran and examined by fluorescence microscopy. Phase contrast and fluorescence views of the same
microscopic fields are shown. The scale bar represents 10 ␮m. D, Determination of lysosomal enzyme activity. The activity of ␣-N-acetylglucosaminidase
was determined with whole-cell lysates of macrophages using a 4-methylumbelliferone-conjugated substrate.
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areas near the nucleus in macrophages before phagocytosis reactions (Fig. 8A). To determine the localization of the tagged TLR4
in macrophages after engulfment, we used apoptotic thymocytes
and latex beads as targets. The results showed that TLR4 was
associated with phagosomes containing either target in a macrophage cell line (Fig. 8B).
INHIBITION OF PHAGOSOME-LYSOSOME FUSION BY TLR4
The Journal of Immunology
2045
TLR2, has been shown to be present in phagosomes when expressed
as an epitope-tagged protein in a mouse macrophage cell line and has
been proposed to discriminate between microbes (37). The presence
of TLR9 in endosomes/lysosomes has also been suggested, because
the macrophage response to unmethylated CpG dinucleotides is abrogated in the presence of inhibitors of endosome/lysosome acidification (38 – 40). The association with and action in phagosomes may
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FIGURE 6. Accelerated fusion between engulfed
cell-containing phagosomes and endosomes/lysosomes in TLR4-deficient macrophages. A, Analysis of
fusion by two assay systems. Wild-type macrophages
whose endosomes/lysosomes had been loaded with
Acridine Orange (assay A) or Lysotracker Red (assay
B) were incubated with unlabeled (assay A) or PKH2labeled (assay B) apoptotic mouse thymocytes and examined by fluorescence/phase contrast microscopy.
Fluorescence and phase contrast views of the same
microscopic fields are shown. Photomicrographs with
the terms B and NIBA indicate examinations by fluorescence microscopy with the wavelengths for excitation/detection of 460 – 490 nm/515⫹ nm and 470 –
490 nm/515–550 nm, respectively. The scale bar
represents 10 ␮m. Arrows and arrowheads indicate
examples of unfused and fused phagosomes, respectively. An asterisk in the top panels points to the nucleus of a macrophage stained light green with Acridine Orange. B, Determination of fusion in
macrophages engulfing various targets. Wild-type (E)
and TLR4-difficient (F) macrophages were incubated
with the indicated target cells or particles and examined for the occurrence of fusion in the two assays at
the indicated time points. The numbers of macrophages containing fused phagosomes in assay A and
of fused phagosomes in assay B were determined and
expressed as a percentage relative to the total macrophages and phagosomes, respectively. The data are the
mean ⫾ SD (n ⫽ 4).
thus be a general feature of TLR. Our results indicated that the absence of TLR4 causes acceleration in the fusion between phagosomes
and endosomes/lysosomes in macrophages engulfing any target cell
examined. This suggests that TLR4 is triggered in phagosomes containing various types of targets with distinct surface structures. It is
therefore difficult at present to predict the mechanism by which phagosomal TLR4 is activated to regulate the fusion.
2046
INHIBITION OF PHAGOSOME-LYSOSOME FUSION BY TLR4
The physiological consequences of TLR4-mediated regulation
of the processing of engulfed apoptotic cells are unclear at the
present time. Engulfed apoptotic cells may have fates other than
degradation in phagocytes. When virus-infected cells are phagocytosed, the components of engulfed cells are processed, resulting
in Ag presentation by dendritic cells (reviewed in Refs. 9 and 10),
or viral genes are transferred to the nuclei of macrophages and
integrated into macrophage DNA (41– 43). However, the mechanisms by which engulfed materials avoid degradation in phagocytes remain to be elucidated. Apart from the clearance of apoptotic cells, the killing of engulfed bacteria seems to be controlled
in phagocytes: some bacteria, when engulfed, inhibit fusion between phagosomes and endosomes/lysosomes and consequently
can survive in macrophages (reviewed in Refs. 44 – 46). Our results indicate that TLR4 acts to delay the fusion of phagosomes
FIGURE 8. Association of TLR4 with phagosomes. RAW264.7 cells transfected with DNA expressing FLAG-tagged TLR4 were immunohistochemically examined for the localization of the tagged
TLR4 using anti-FLAG Ab before (A) and after (B)
phagocytosis reactions. Phase contrast and fluorescence views of the same microscopic fields are shown.
The scale bars represent 10 ␮m. A, Examples of cells
showing surface (top) and perinuclear (bottom) distribution of the tagged TLR4 are shown. B, Examples of
engulfed thymocytes and latex beads are indicated by
an asterisk and an arrowhead, respectively. The bottom panels are magnified views of the corresponding
top panels.
with endosomes/lysosomes. This leads us to speculate that TLR4
is involved in the above-mentioned phenomena by prolonging the
lifetime of engulfed materials in macrophages. We previously
showed that apoptosis-dependent phagocytosis of influenza virus-infected cells by macrophages leads to inhibition of the release of viral
progeny into the culture medium (7). Results from our on-going experiments have indicated that the level of influenza virus production
was almost equal when virus-infected cells were incubated with wildtype and TLR4-deficient macrophages (our unpublished observation).
Another event that might be influenced by TLR4 deficiency is engulfment-dependent changes in gene expression in phagocytes (47,
48); for example, the expression patterns of inflammation-related cytokines change in macrophages after the engulfment of apoptotic cells
(49 –51). Regulation of the fusion between apoptotic cell-containing
phagosomes and endosomes/lysosomes might be involved in one or a
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FIGURE 7. Independence of TLR4 regulation of the processing of phagosomes from NF-␬B. A, Detection of NF-␬B activity in nuclear extracts of
macrophages. Wild-type macrophages were incubated with apoptotic mouse thymocytes or LPS for the indicated lengths of time. Nuclear extracts were
prepared from those cells and subjected to EMSA with an NF-␬B probe. A digital image of a polyacrylamide gel in which the EMSA reactions were
resolved is shown. The two right lanes contained reaction mixtures treated with Ab recognizing NF-␬B p65 subunit or C/EBP-␤. The positions of free
probe, NF-␬B/probe complex, and signal shifted by Ab (supershift) are indicated. B, Effect of an inhibitor of nuclear translocation of NF-␬B on macrophage
phagocytosis. Phagocytosis of apoptotic mouse thymocytes by wild-type macrophages was conducted in the presence and the absence of DHMEQ, which
was added 2 h before and continued to be present during the reaction. The data are the mean ⫾ SD (n ⫽ 4). C, Effect of LPS on macrophage phagocytosis.
Phagocytosis of apoptotic mouse thymocytes by wild-type macrophages was conducted in the presence and the absence of LPS, which was added 1 h before
and continued to be present during the reaction. The data are the mean ⫾ SD (n ⫽ 4).
The Journal of Immunology
number of the above-mentioned events; if so, TLR4 might contribute
to the innate immune response in one or more ways, resulting in
enhanced maintenance of tissue homeostasis.
Acknowledgments
We thank H. Fujita, Y. Tanaka, and M. Himeno for anti-LGP85 and anticathepsin D Ab; Y. Nagai and K. Miyake for pEFBOS/mouse
TLR4flaghis; K. Umezawa for DHMEQ, D. Takayama for anti-mouse
Thy1 mAb G7; and Y. Osumi, N. Mizushima, S. Ohkuma, K. Itoh,
H. Fujita, and H. Nakanishi for advice.
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