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Journal of Virology, October 2000, p. 9240-9244, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Phosphatidylserine-Mediated Phagocytosis of
Influenza A Virus-Infected Cells by Mouse Peritoneal
Macrophages
Akiko
Shiratsuchi,1
Masako
Kaido,1
Takenori
Takizawa,2 and
Yoshinobu
Nakanishi1,*
Graduate School of Natural Science and
Technology, Kanazawa University, Kanazawa, Ishikawa
920-0934,1 and Institute for
Developmental Research, Aichi Human Service Center, Kasugai, Aichi
480-0392,2 Japan
Received 18 April 2000/Accepted 5 July 2000
 |
ABSTRACT |
Influenza virus induces apoptosis in cultured cell lines as well as
in animal tissues. HeLa cells were infected with influenza virus
A/Udon/72 (H3N2) under conditions resulting in almost 100% infection.
Such cells underwent typical caspase-dependent apoptosis and were
efficiently phagocytosed by macrophages prepared from peritoneal fluids
of thioglycolate-treated mice. The membrane phospholipid
phosphatidylserine appeared on the surfaces of virus-infected cells at
around the time efficient phagocytosis became detectable. In fact, the
phagocytosis was almost completely inhibited in the presence of
liposomes containing phosphatidylserine, which did not influence the
antibody-dependent uptake of zymosan particles by the same macrophages.
These results indicate that macrophages phagocytose influenza
virus-infected HeLa cells in a manner mediated by phosphatidylserine
that appears on the surfaces of infected cells during the process of apoptosis.
| |
INTRODUCTION |
Many viruses induce apoptotic death
in host cells, but the physiological meaning of this phenomenon is not
yet understood (9, 27). HeLa cells infected with influenza A
virus undergo Fas/Fas ligand-mediated and caspase-dependent apoptosis
(7, 25, 28), but the virus appears to replicate normally and
virus progeny are released into the culture medium (26).
Cells undergoing apoptosis are in general rapidly and selectively
engulfed by phagocytes (3, 29), and this presumably prevents
inflammation that would otherwise be caused by the noxious contents of
dead cells (14, 16, 17). It could thus be reasonably
expected that influenza virus-infected cells would be susceptible to
apoptosis-dependent phagocytosis.
Recently, it was reported that phagocytosis of apoptotic cells leads to
antigen presentation to lymphocytes; dendritic cells that phagocytosed
influenza virus-infected cells undergoing apoptosis stimulated
CD8+ T cells (1, 2). It was shown, on the other
hand, that phagocytosis of influenza virus-infected cells by
macrophages led to elimination of the virus (8). In both
cases, virus-infected cells were phagocytosed depending on the
occurrence of apoptosis (2, 8). These results suggest that
apoptosis of influenza virus-infected cells protects the organism from
viral invasion in a dual manner. Apoptosing cells expose a phagocytic
marker(s) on their surfaces, and phagocytes recognize this marker and
use a specific receptor(s) to engulf the cells presenting this marker
(14, 16, 17). As a first step toward an understanding of the
molecular basis of the phagocytosis of influenza virus-infected cells,
we searched for the marker molecule(s) responsible for the recognition
of influenza virus-infected cells by macrophages.
| |
MATERIALS AND METHODS |
Infection and growth of influenza virus in HeLa cells.
HeLa
S3 cells were maintained in Eagle's minimal essential medium
containing 10% fetal bovine serum at 37°C in 5% CO2.
HeLa cells were infected with a wild-type strain of influenza A/Udon/72 (H3N2) virus, SP626, at a multiplicity of infection of two, as described previously (7, 28). Virus growth was monitored by
either an immunohistochemical analysis or a plaque assay
(24). For immunohistochemistry, influenza virus-infected
HeLa cells were maintained in poly-D-Lys-coated culture
containers, fixed, and permeabilized, as described previously
(8). The cells were treated with an anti-influenza virus
antiserum which recognizes NP, M1, HA, and NA (19) and then
with a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit
immunoglobulin G (IgG) antibody (Immunotech, Marseilles, France) and
examined by fluorescence and phase-contrast microscopy (BX50
microscope; Olympus, Tokyo, Japan). The amount of virus released into
the culture medium was determined by a plaque assay using MDCK cells
and expressed as PFU as described previously (25).
Apoptosis analysis.
Cell viability and chromatin
condensation were analyzed under a microscope after staining cells with
trypan blue and Hoechst 33342, respectively. Translocation of the
membrane phospholipid phosphatidylserine (PS) from the cytoplasmic to
the exoplasmic leaflet of the plasma membrane was determined by flow
cytometry using annexin V, which specifically binds to PS, as described previously (10, 12). In brief, cells were treated with
FITC-labeled annexin V (Bender MedSystems, Vienna, Austria) and
propidium iodide, a membrane-impermeative fluorochrome, and analyzed in
a flow cytometer (EPICS-XL; Coulter, Hialeah, Fla.). The cells that
were less intensely stained with propidium iodide and thus impermeable
by annexin V were gated and analyzed for the amount of bound
FITC-annexin V. To inhibit apoptosis, z-VAD-fmk (23)
(Peptide Institute, Osaka, Japan), an inhibitor of caspases, was added
to the medium when virus-infected cells were put into the culture dishes.
Macrophage preparation and phagocytosis assay.
Macrophages
were isolated from the peritoneal cavities of thioglycolate-treated
BDF1 mice and maintained in RPMI 1640 containing 10% fetal
bovine serum at 37°C until use, as described previously (4,
21). The phagocytosis assay was performed essentially as
described previously (20). Briefly, HeLa cells were labeled with biotin (NHS-LS-Biotin; Pierce, Rockford, Ill.), mixed with macrophages (at a ratio of five target cells to one macrophage), and
incubated at 37°C for 2 h. The mixture was washed by pipetting with phosphate-buffered saline (PBS) and then with trypsin (0.5 µg/ml) to remove HeLa cells free from or lightly attached to
macrophages. The remaining cells were further fixed with PBS containing
2% paraformaldehyde, 0.5% glutaraldehyde, and 0.05% Triton X-100 and
then supplemented with FITC-conjugated avidin (fluorescein-avidin D;
Vector, Burlingame, Calif.). The number of macrophages containing engulfed cells was determined using fluorescence and phase-contrast microscopy and expressed relative to the total number of macrophages; this ratio was termed the phagocytic index. Zymosan particles (Sigma,
St. Louis, Mo.) were swollen in water at 100°C for 1 h and
washed with PBS. The particles were then labeled with
5-carboxyfluorescein, succinimidyl ester (Molecular Probes, Eugene,
Oreg.), and incubated with mouse IgG (Zymed, San Francisco, Calif.).
The fluorescein-labeled and opsonized zymosan particles were added to
macrophages that had been plated on coverslips with serum-free medium.
The mixture was kept on ice for 10 min and then incubated at 37°C for
1 h. Unincorporated particles were washed out by pipetting with
PBS, and the macrophages were fixed. The phagocytic index was
determined as described above. The means and standard deviations (SDs)
for typical examples from at least three independent experiments are presented.
Liposome preparation.
Phospholipids (Avanti Polar Lipids,
Alabaster, Ala.) were dried as films, suspended in PBS, and sonicated
(20). Liposomes were formed using either phosphatidylcholine
only (PC liposomes) or a combination of phosphatidylcholine and PS at a
molar ratio of 7:3 (PS liposomes).
| |
RESULTS |
Virus growth and apoptosis in influenza virus-infected HeLa
cells.
When HeLa cells infected with a wild-type strain of
influenza virus for various periods were subjected to an
immunohistochemical analysis using an antiserum against virions, the
cells were almost 100% positive at 6 h after infection (Fig.
1A, left panels), and the positivity
continued for the next 18 h (data not shown). The same antibody
did not react with uninfected cells (Fig. 1A, right panels). The virus
titer in the culture medium, which was determined by a plaque assay
using MDCK cells, increased as the time of culturing of the infected
cells increased (Fig. 1B). The titer started to increase at 9 h
and reached a plateau at 12 h. These results indicate that near
100% infection was achieved and that virus release began at
approximately 9 h postinfection under these conditions.
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FIG. 1.
Growth of influenza virus in HeLa cells. (A)
Immunohistochemical analysis of virus-infected HeLa cells with an
antibody raised against virions. HeLa cells at 6 h postinfection
(left) and mock-infected cells (right) were treated with the primary
antibody followed by the addition of a fluorescence-labeled secondary
antibody and examined under a fluorescence and a phase-contrast
microscope. Fluorescence (top) and phase-contrast (bottom) views are
shown. Bar = 50 µm. (B) Time course of virus growth. The virus
titer in the culture medium was determined by a plaque assay. A typical
example from three independent experiments is shown with the means and
SDs.
|
|
We then determined the time course of apoptosis in influenza
virus-infected cells in terms of increases in plasma membrane
permeability and in the number of cells with condensed chromatin.
These
changes became evident almost simultaneously after 9 h of
infection (Fig.
2).
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FIG. 2.
Influenza virus-induced apoptosis of HeLa cells. HeLa
cells infected with influenza virus for the indicated periods were
analyzed for cell viability and chromatin condensation. The percentages
of cells with impermeable plasma membranes and condensed chromatin are
presented.
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Phagocytosis of influenza virus-infected HeLa cells by mouse
peritoneal macrophages.
HeLa cells infected with influenza virus
for 24 h were labeled with biotin and mixed with mouse peritoneal
macrophages. The mixture was further cultured for 2 h, washed,
fixed, permeabilized, and supplemented with FITC-avidin. When the
reacted cells were examined by fluorescence and phase-contrast
microscopy, many fluorescent particles were detected in the cytoplasms
of macrophages, but control mock-infected cells were not significantly
engulfed by macrophages (data not shown). Quantitative analysis of the
phagocytosis reaction revealed that HeLa cells became susceptible to
phagocytosis by macrophages upon infection with influenza virus (Fig.
3A). In order to more directly examine
whether phagocytosed cells were infected with influenza virus, after
the phagocytosis reaction macrophages were simultaneously treated with
Texas red-labeled avidin and an anti-influenza virus antibody followed
by the addition of an FITC-labeled secondary antibody and examined by
fluorescence and phase-contrast microscopy. Most of the engulfed HeLa
cells, stained in red, were positive for the antibody (Fig. 3B),
indicating that influenza virus-infected cells were selectively
phagocytosed by macrophages. We next determined the extent of
phagocytosis, using HeLa cells that had been infected with influenza
virus for various periods. Phagocytosis became evident at significant
levels at 9 h, and the phagocytic index continued to increase
thereafter (Fig. 3C). This indicates that virus release, apoptosis
induction, and phagocytosis by macrophages occurred with similar time
courses during infection.
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FIG. 3.
Phagocytosis of influenza virus-infected HeLa cells by
mouse peritoneal macrophages. (A) Cells infected with virus for 18 h and mock-infected cells were subjected to a phagocytosis assay. The
means and SDs are shown. (B) Immunohistochemical analysis of
macrophages after the phagocytosis reaction with the anti-influenza
virus antibody. All HeLa cells (18 h postinfection) used were marked by
Texas red (red), and virus-infected cells were detected with an
FITC-conjugated secondary antibody (green). Top left, FITC signal from
virus-infected cells; top right, Texas red signal from all HeLa cells;
bottom left, merged view of Texas red and FITC signals; bottom right,
phase-contrast view. Bar = 10 µm. (C) Time course of
phagocytosis reaction. HeLa cells infected with influenza virus for the
indicated periods were subjected to a phagocytosis assay.
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Involvement of PS in phagocytosis of virus-infected cells.
Since phagocytosis of influenza virus-infected HeLa cells depends on
the occurrence of apoptosis (8), the infected cells are
likely to possess a phagocytosis marker(s) that makes apoptotic cells
recognizable by phagocytes. Previous experiments showed that the
phagocytosis marker PS, a phospholipid which is normally restricted to
the inner leaflet of the membrane bilayer, translocates to the outer
leaflet and is exposed to the surfaces of influenza virus-infected HeLa
cells (7). Such PS externalization occurred about 9 h
after virus infection (Fig. 4A), at which
time the extent of phagocytosis increased greatly (Fig. 3C). We thus
hypothesized that PS serves as a phagocytosis marker for influenza
virus-infected HeLa cells.
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FIG. 4.
Caspase-dependent externalization of PS in influenza
virus-infected HeLa cells. (A) HeLa cells infected with influenza virus
for the indicated periods were analyzed for PS externalization in a
flow cytometer. Cells less intensely stained with propidium iodide
(bottom area in the left panels) were analyzed for the binding of
annexin V (right panels). Thick lines, mock-infected cells; thin lines,
virus-infected cells. Numbers in the left panels indicate the
percentages of cells in the corresponding areas. (B) HeLa cells
infected with virus for 9 h in the absence or presence of
z-VAD-fmk were analyzed for PS externalization. Vertical dotted lines
indicate the mean fluorescence in the analysis of mock-infected
cells.
|
|
We first examined whether PS externalization requires activated
caspases. To test this, influenza virus infection was done
in the
presence of z-VAD-fmk, and the amount of PS on the cell
surface was
determined by flow cytometry. This inhibitor does
not affect the growth
of influenza virus (
26). The results clearly
showed that the
increase in the number of cells with externalized
PS which was observed
upon virus infection was almost completely
abrogated by the addition of
the inhibitor (Fig.
4B). It was thus
concluded that PS externalization
in influenza virus-infected
HeLa cells depends on apoptosis and is an
event downstream of
the caspase
cascade.
In order to examine whether PS externalized on the surfaces of
virus-infected cells is recognized by macrophages, we conducted
a
phagocytosis assay in the presence of PS liposomes using HeLa
cells
infected with virus for various periods. PS liposomes significantly
inhibited phagocytosis at all times examined, whereas PC liposomes
showed a minimal effect (Fig.
5A). When
the liposomes were added
at various concentrations, PS liposomes
inhibited phagocytosis
in a dose-dependent manner, while PC liposomes
did not show an
inhibitory effect at any concentration (Fig.
5B). In
contrast,
macrophage uptake of IgG-coated zymosan particles, which is
presumably
mediated through Fc/Fc receptor interaction, was not
affected
by PS liposomes (Fig.
5C). This shows that inhibition of
phagocytosis
of virus-infected cells does not reflect a nonspecific
effect
of liposomes on macrophage action. All of these results
collectively
indicated that phagocytosis of influenza virus-infected
HeLa cells
by macrophages was mediated by PS which was exposed on the
surfaces
of HeLa cells during the process of apoptosis.
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FIG. 5.
Effect of liposomes on phagocytosis of influenza
virus-infected HeLa cells. (A) HeLa cells infected with virus for the
indicated periods were subjected to a phagocytosis assay in the
presence of PS liposomes or PC liposomes (1 mM). (B) Liposomes were
added at various concentrations in the phagocytosis reaction using HeLa
cells infected for 24 h. The extent of phagocytosis is shown
relative to that for a control reaction with no added liposomes, which
was considered 100%. (C) Phagocytosis of opsonized zymosan particles
by macrophages was conducted in the presence or absence of PS
liposomes. The extent of phagocytosis is shown relative to that for a
control reaction with no added liposomes, which was considered 100%.
The means of the phagocytic index in control reactions were 2.5 (9 h),
6.7 (12 h), 10 (18 h), 11 (24 h [A]), 14 (24 h [B]), and 34 (C).
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| |
DISCUSSION |
Influenza virus-infected cells are engulfed by phagocytes, such as
dendritic cells (1, 2) and macrophages (2, 8), in
an apoptosis-dependent manner. Apoptosing cells are generally recognized by phagocytes through marker molecules, which appear on cell
surfaces upon apoptosis induction (14, 16, 17). Therefore,
it was reasonable to expect that apoptosing influenza virus-infected
cells possess such a marker. The results of this study revealed that
PS, the best-characterized phagocytosis marker (6, 11), is
the one that makes macrophages recognize and engulf influenza
virus-infected cells. PS, which is localized at the cytoplasmic side of
the membrane bilayer in normal cells (30), translocated to
cell surfaces during the apoptosis pathway in influenza virus-infected
cells and served as a phagocytosis marker. Macrophages are likely to
recognize influenza virus-infected cells using a presumed PS receptor,
and one such molecule has recently been identified (5). The
presence of a phagocytosis-inducing PS receptor has also been reported
for other phagocytes, such as testicular Sertoli cells (22)
and vascular endothelial cells (13). Albert et al.
(2) suggested that dendritic cells recognize influenza
virus-infected monocytes using 
V
5 integrin and CD36, the latter
of which binds to PS (15). Although the involvement of PS in
the recognition between the two cell types has not been examined, CD36
could be a PS receptor that induces dendritic cell phagocytosis.
The extent of phagocytosis continued to increase even after PS
externalization was completed at 12 h postinfection, and
phagocytosis at all time points was almost completely inhibited by the
addition of PS liposomes. This indicates that the exposure of PS is
necessary but not sufficient for efficient phagocytosis of influenza
virus-infected cells. We presume the presence of another molecule which
is involved in recognition of virus-infected cells by macrophages, most
probably in cooperation with PS. Some viral protein(s) could be such a molecule, since an antihemagglutinin antibody inhibited the binding of
virus-infected cells to macrophages (18). We previously
showed that cells exposing PS independent of apoptosis are
phagocytosed, but only inefficiently, in a PS-mediated manner
(21). It is thus possible that a candidate molecule(s) gains
its function upon apoptosis induction. Identification of such a
molecule(s) is important for fully understanding the molecular basis of
phagocytosis, not only of influenza virus-infected cells but also of
apoptotic cells in general.
Phagocytosis of influenza virus-infected cells leads to inhibition of
virus growth (9). We showed here that influenza
virus-treated cells became susceptible to macrophage phagocytosis at an
early stage of infection. It is thus anticipated that phagocytosis of influenza virus-infected cells plays a role in the initial defense against viral invasion. Since phagocytosis of influenza virus-infected cells by dendritic cells leads to antigen presentation to
CD8+ T lymphocytes (1, 2), apoptosis-dependent
phagocytosis of virus-infected cells may protect the organism from
influenza virus in two different ways. Further experiments using
experimental animals will be necessary to examine whether such a host
defense system really functions in vivo.
| |
ACKNOWLEDGMENTS |
We thank K. Shimizu for the anti-influenza virus antiserum.
This study was supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, Sports, and Culture of Japan; by a
grant from the Organized Research Combination System of the Science and
Technology Agency of Japan; and by a grant from the Uehara Memorial Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Graduate School
of Natural Science and Technology, Kanazawa University, Takara-machi, Kanazawa, Ishikawa 920-0934, Japan. Phone: 81-76-234-4481. Fax: 81-76-234-4480. E-mail:
nakanaka{at}kenroku.kanazawa-u.ac.jp.
| |
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Journal of Virology, October 2000, p. 9240-9244, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Hashimoto, Y., Moki, T., Takizawa, T., Shiratsuchi, A., Nakanishi, Y.
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Abstract
Introduction
