Originally published In Press as doi:10.1074/jbc.M201074200 on March 7, 2002
J. Biol. Chem., Vol. 277, Issue 20, 18222-18228, May 17, 2002
Role of Phosphatidylserine Exposure and Sugar Chain Desialylation
at the Surface of Influenza Virus-infected Cells in Efficient
Phagocytosis by Macrophages*
Yuichi
Watanabe
,
Akiko
Shiratsuchi,
Kazufumi
Shimizu§,
Takenori
Takizawa¶, and
Yoshinobu
Nakanishi
From the Graduate School of Medical Science, Kanazawa
University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan,
§ School of Medicine, Nihon University, Itabashi-ku, Tokyo
173-8610, Japan, and ¶ Institute for Developmental Research,
Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan
Received for publication, February 1, 2002, and in revised form, February 28, 2002
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ABSTRACT |
HeLa cells infected with influenza A virus
undergo typical caspase-dependent apoptosis and are
efficiently phagocytosed by mouse peritoneal macrophages in a manner
mediated by the membrane phospholipid phosphatidylserine, which is
translocated to the surface of virus-infected cells during apoptosis.
However, the extent of phagocytosis is not always parallel with the
level of phosphatidylserine externalization. Here we examined the
involvement of influenza virus neuraminidase (NA) in efficient
phagocytosis of virus-infected cells. HeLa cells infected with an
influenza virus strain expressing temperature-sensitive NA underwent
apoptosis and produced viral proteins, including the defective NA, at a non-permissive temperature to almost the same extent as cells infected
with the wild-type virus. The cells were, however, phagocytosed by
macrophages with reduced efficiency. In addition, phagocytosis of cells
infected with the wild-type virus was severely inhibited when the cells
had been maintained in the presence of the NA inhibitor zanamivir. On
the other hand, the binding of sialic acid-recognizing lectins to the
cell surface declined after infection with the wild-type virus. The
decrease in the extent of lectin binding was greatly attenuated when
cells were infected with the mutant virus or when wild-type
virus-infected cells were maintained in the presence of zanamivir.
These results indicate that sugar chains are desialylated by NA at the
surface of virus-infected cells. We conclude that the presence of both
phosphatidylserine and asialoglycomoieties on the cell surface is
required for efficient phagocytosis of influenza virus-infected cells
by macrophages.
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INTRODUCTION |
Cells undergoing apoptosis are selectively and rapidly eliminated
from the organism by phagocytosis (1, 2), and this process contributes
to maintaining tissue homeostasis (3, 4). Phagocytes such as
macrophages bind to apoptotic cells by recognizing phagocytosis marker
molecules that are expressed on the surface of target cells (4, 5); the
membrane phospholipid phosphatidylserine (PS)1 is the best
characterized phagocytosis marker (6-9). PS is normally restricted to
the inner leaflet of the membrane bilayer (10, 11) but translocates to
the outer leaflet and is exposed to the cell surface during apoptosis
by as yet unknown mechanisms (6, 9). Although the externalization of PS
seems to occur in many types of apoptotic cells, the presence of other
phagocytosis markers that may cooperate with PS in phagocyte
recognition of apoptotic cells has been proposed (12-15).
Many viruses induce apoptosis in host cells, but the physiological
consequences of this phenomenon are not fully understood (16, 17).
Cells infected with influenza A virus undergo typical apoptosis that
has been considered to be caused by Fas ligand and Fas, the expression
of which is simultaneously stimulated upon viral infection (18-21).
The involvement of viral genome-encoded proteins such as neuraminidase
(NA) (22), nonstructural protein 1 (23, 24), and the newly discovered
protein PB1-F2 (25) in the regulation of apoptosis in influenza
virus-infected cells has also been reported, although the mode of
action of nonstructural protein 1 is controversial. Influenza
virus-infected HeLa cells become susceptible to
apoptosis-dependent phagocytosis by mouse peritoneal
macrophages, and the phagocytic clearance of virus-infected cells
appears to inhibit the expansion of viral infection (26). It can thus
reasonably be presumed that apoptosis of influenza virus-infected cells
leads to elimination of the virus from the organism. We previously
showed that macrophages recognized PS that was expressed at the surface
of influenza virus-infected cells during apoptosis, but the extent of
phagocytosis continued to increase even after the level of cell surface
PS reached a maximum (27). This indicates that PS is not solely
responsible for the recognition of influenza virus-infected cells by
macrophages. We therefore decided to search for other surface change(s)
needed for efficient phagocytosis of virus-infected cells. Influenza virus-infected cells express viral envelope proteins on their surface,
and these proteins might participate in the recognition and
phagocytosis of the cells by macrophages. We first examined the
involvement of NA, one of the envelope proteins, because NA inhibitors
and a mutant influenza virus strain with defective NA were available.
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EXPERIMENTAL PROCEDURES |
Virus Strains and Infection--
The strains of influenza A
virus used in this study were derivatives of influenza A/Udorn/72
(H3N2) virus, clones SP626 (wild type) and ICRC282 (a
temperature-sensitive (ts) mutant). Clone ICRC282, which produces ts NA
(28, 29), was obtained by mutagenizing the wild-type virus with the
acridine-based compound ICR 191, as described previously (30, 31). For
virus adsorption, HeLa S3 cells maintained in Eagle's minimal
essential medium containing 10% fetal bovine serum at 37 °C in 5%
CO2, 95% air were incubated with phosphate-buffered saline
(PBS) containing either the wild-type or the ts mutant virus at a
multiplicity of infection of one at room temperature for 30 min, as
described previously (20, 21). For control "mock" infection, cells
were similarly treated with PBS alone. Those cells were then cultured
at 40 °C, a non-permissive temperature for the ts NA, in 5%
CO2, 95% air and used for further analyses. In some
experiments, cells infected with the wild-type influenza virus were
cultured at 37 °C in 5% CO2, 95% air in the presence
of 10 µM zanamivir
(4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) (a gift from GlaxoSmithKline), a specific inhibitor of NA (32),
and then further analyzed. All culturing was done in the absence of
trypsin to avoid secondary infection by the replicated virus.
Determination of Virus Growth--
The growth of influenza virus
in HeLa cells was monitored by examining the production of viral
proteins by immunohistochemistry or Western blotting. For
immunohistochemistry, virus-infected cells were maintained in
poly-D-Lys-coated culture containers, fixed, and
permeabilized as described previously (26). The cells were treated with
an anti-influenza virus antiserum, which recognizes nucleoprotein (NP),
matrix protein-1, hemagglutinin (HA), and NA (33), and then with a
fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin
G antibody (Vector, Burlingame, CA). The cells were examined by
fluorescence and phase-contrast microscopy (BX50 microscope; Olympus,
Tokyo, Japan). For Western blotting, virus-infected cells were lysed
with a buffer containing 62.5 mM Tris-HCl (pH 6.8), 2.5%
SDS, and 2.5% 2-mercaptoethanol, and the lysates were separated by
electrophoresis on a 15% SDS-polyacrylamide gel. The proteins were
electrophoretically transferred onto a polyvinylidene difluoride
membrane (Immobilon P; Millipore, Bedford, MA). The membrane was
blocked with 5% dry skim milk, incubated with the anti-influenza
antiserum in a buffer consisting of 20 mM Tris-HCl (pH
7.5), 0.15 M NaCl, 0.5% Tween 20, and 5% dry skim milk,
washed, reacted with an alkaline phosphatase-conjugated anti-rabbit
immunoglobulin G antibody (Bio-Rad), and subjected to a
chemiluminescence reaction using the Immun-Star system (Bio-Rad). The
signals were assessed using Fluor-S MAX (Bio-Rad).
Apoptosis Analysis--
The integrity of the plasma membrane and
the occurrence of chromatin condensation were determined by examination
under a microscope after staining cells with trypan blue and Hoechst
33342, respectively (27, 34). The number of cells negative for trypan
blue staining or having condensed chromatin was determined and
expressed (in percentage) relative to the total number of cells.
Translocation of PS from the cytoplasmic to the exoplasmic leaflet of
the plasma membrane was assessed by flow cytometry using annexin V,
which specifically binds to PS, as described previously (35, 36). In
brief, cells were treated with FITC- or 5-carboxyfluorescein-labeled annexin V and propidium iodide, a membrane-impermeable fluorochrome, and analyzed in a flow cytometer (EPICS-XL; Coulter, Hialeah, FL). The
cells that were less intensely stained with propidium iodide and thus
impermeable to annexin V were gated and analyzed for the amount of
bound annexin V.
NA Assay--
The activity of NA was determined using a
colorimetric assay with fetuin as the substrate (37, 38). In brief,
virus- or mock-infected HeLa cells (2 × 106) were
suspended in 40 mM acetate buffer (pH 5.6) containing 4 mg/ml fetuin (Sigma) and incubated at 37 °C for 1 h.
Sialic acids released from fetuin were brought to the coloring
reaction, and their amounts were determined by measuring the
A549.
Analysis of Glycosylated Proteins--
Either the wild-type or
the NA mutant influenza virus was adsorbed to 1 × 106
HeLa cells at a multiplicity of infection of 10 as described above, and
the infected cells were maintained at 40 °C for 6 h. They were
then incubated with 3H-labeled glucosamine
(D-[1,6-3H]glucosamine hydrochloride, 49 Ci/mmol; PerkinElmer Life Sciences) (40 µCi/ml) in serum-free
medium at 40 °C for 30 min. The cells were harvested using a cell
scraper, suspended in the buffer used to lyse cells for Western
blotting, heated at 50 °C for 10 min, lysed by passage through a
26-gauge needle 15 times, and electrophoresed on a 10%
SDS-polyacrylamide gel. The gel was fixed with a solution of 20%
methanol and 5% acetic acid, treated with ENHANCE (PerkinElmer Life
Sciences), dried, and autoradiographed.
Macrophage Preparation and Phagocytosis Assay--
Macrophages
were isolated from the peritoneal cavity of thioglycolate-treated
BDF1 mice (females, 8-12 weeks old) and maintained in RPMI
1640 medium containing 10% heat-inactivated fetal bovine serum at
37 °C until use, as described previously (34, 39). The phagocytosis
assay was performed essentially as described (27, 40). Briefly, HeLa
cells infected with influenza virus were labeled with biotin
(NHS-LC-Biotin; Pierce), mixed with macrophages (at a ratio of three
target cells to one macrophage), and incubated at 37 °C for 2 h. The cell mixture was then treated with trypsin (0.5 µg/ml) to
remove HeLa cells free from or lightly attached to macrophages. The
remaining cells were fixed, permeabilized, and supplemented with
FITC-conjugated avidin (fluorescein-avidin D; Vector Laboratories). The
number of macrophages containing engulfed cells was determined by
fluorescence and phase-contrast microscopy and expressed relative to
the total number of macrophages; this ratio (in percentage) was termed
the phagocytic index. The means ± S. D. of a typical example
from at least three independent experiments are presented. Statistical
analysis was performed by the Student's t test.
Liposome Preparation--
Phospholipids (Avanti Polar Lipids,
Alabaster, AL) were dried as films, suspended in PBS, and
sonicated (40). 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).
Lectin Binding Assay--
Either the wild-type or the NA mutant
influenza virus was adsorbed to 2 × 106 HeLa cells at
a multiplicity of infection of 1 as described above, and the infected
cells were maintained in serum-free medium at 40 °C for 16 h.
To examine the effects of zanamivir, the entire process of virus
adsorption (wild type) and culturing (at 37 °C for 20 h) of the
virus-infected cells was done in the presence of the drug (10 µM). The cells were suspended in PBS containing FITC-labeled lectin (Honen, Tokyo, Japan) (20-30 µg/ml) and
propidium iodide and incubated at room temperature for 30 min. The
samples were then washed with PBS and analyzed by flow cytometry. The cells less intensely stained with propidium iodide were gated and
analyzed for the amount of bound FITC-lectins. The lectins used in this
study were wheat germ agglutinin, which recognizes (GlcNAc)2, (GlcNAc)3, and Neu5Ac; Maackia
amurensis lectin, which recognizes Neu5Ac
2-3Gal
1-4GlcNAc;
and Sambucus sieboldiana lectin, which recognizes
Neu5Ac2-3Gal and Neu5Ac2-6GalNAc.
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RESULTS |
Characterization of Cells Infected with Mutant Influenza
Virus--
HeLa cells that had been infected with either the wild-type
or the ts mutant influenza virus were cultured at a non-permissive temperature (40 °C) for various lengths of time and assayed for NA
activity (Fig. 1A). The NA
activity in cells infected with the wild-type virus continued to
increase during the culture reaching a maximum level at 24 h. In
contrast, cells infected with the mutant virus showed only the basal
level of activity throughout the culture period, as did mock-infected
cells (data not shown). However, we found no significant difference in
the NA activity between cells infected with the wild-type and the
mutant viruses when they were cultured at 34 °C, a permissive
temperature (data not shown). All these results agreed well with our
previous observations (28, 29).
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Fig. 1.
Detection of NA activity and viral proteins
in influenza virus-infected cells. A, HeLa cells
infected with either the wild-type or the ts mutant virus were cultured
at 40 °C (a non-permissive temperature) for the indicated periods
and assayed for NA activity. B, virus-infected cells were
maintained at either a non-permissive (40 °C) or a permissive
(34 °C) temperature for 16 h and analyzed by immunofluorescence
using an anti-influenza virus antiserum. Phase-contrast and
fluorescence views of the same fields are shown. Bar = 50 µm. C, total cell lysates (5 µg of protein) prepared
from virus-infected cells that had been cultured at 40 °C for
16 h were analyzed by Western blotting using the anti-influenza
virus antiserum. The positions of molecular mass markers
(left) and viral proteins (right) are indicated.
M1, matrix protein 1. D, glycoproteins in
virus-infected cells were labeled with [3H]glucosamine
and analyzed by SDS-polyacrylamide gel electrophoresis as described
under "Experimental Procedures." An autoradiogram of the gel is
shown. The positions of HA and NA together with molecular mass markers
are indicated.
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We then used immunohistochemistry and Western blotting to examine the
growth of the mutant virus by detecting viral proteins. When cells
cultured at 40 °C for 16 h after virus adsorption were examined
immunohistochemically with the anti-influenza virus antiserum (Fig.
1B), the proportions of positive cells were 0, 45 ± 6.4, and 86 ± 4.2% with mock-, wild-type virus-, and mutant
virus-infected cells, respectively. The proportion of positive cells
was always larger with the mutant virus than with the wild-type virus.
The signals were almost exclusively localized in the nucleus, whereas viral proteins seemed to be located in the cytoplasm when culturing was
carried out at 34 °C. Virus-infected cells cultured at 34 °C
showed nuclear signals at earlier times (data not shown). This indicated that culturing virus-infected cells at 40 °C delays the
virus propagation compared with that in cells cultured at 34 °C.
When lysates prepared from cells infected with the two virus strains
were subjected to Western blotting, the overall pattern and intensity
of the signals, which were absent in the lysates of mock-infected
cells, were not significantly different between the two types of lysate
(Fig. 1C). All the above results indicate that the mutant
virus with the ts NA grows normally in infected HeLa cells at a
non-permissive temperature. Unfortunately, we were unable to identify
the signal derived from NA, probably because its expression
level was much lower than that of closely migrating NP. We thus
radiolabeled glycoproteins to detect the glycoprotein NA,
distinguishing it from unglycosylated NP. HeLa cells infected with
the wild-type or the mutant influenza virus were incubated with
3H-labeled glucosamine, lysed, and subjected to
SDS-polyacrylamide gel electrophoresis followed by autoradiography
(Fig. 1D). Upon infection with influenza virus, the
synthesis of cellular proteins is repressed, and proteins encoded by
the viral genome are predominantly expressed. Two distinct signals with
slower mobility were detected in each lysate, and these signals were
considered to correspond to viral glycoproteins HA and NA based on
their estimated molecular masses. The intensity of the signal derived
from NA was almost equal in the lysates prepared from cells infected
with either the wild-type or the mutant virus. This indicates that the
ts NA is produced and glycosylated in HeLa cells as efficiently as the
wild-type NA even at a non-permissive temperature and thus suggests
that the defective NA is present on the surface of mutant virus-infected cells.
We next examined the occurrence of apoptosis in cells that had been
infected with the mutant virus and maintained at a non-permissive temperature (Fig. 2). Cells infected with
either the wild-type or the NA mutant virus showed similar extents of
loss of plasma membrane integrity (panel A) and condensation
of chromatin (panel B). On the other hand, the level of PS
externalization in the mutant virus-infected cells was slightly higher
than in cells infected with the wild-type virus (panel C).
These results indicate that the mutant and the wild-type influenza
viruses propagate and induce apoptosis almost equally in HeLa cells at
40 °C.
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Fig. 2.
Induction of apoptosis in influenza
virus-infected cells. HeLa cells infected with the wild-type or
the mutant virus were cultured at a non-permissive temperature
(40 °C) and analyzed for the occurrence of apoptosis. A,
loss of plasma membrane integrity. The proportion of cells not stained
with trypan blue is shown as the mean ± S.D. (n = 3). B, chromatin condensation. The cells were treated with
Hoechst 33342, and the proportion of cells with condensed chromatin is
shown as the mean ± S.D. (n = 3). C,
PS externalization. Cells were cultured for 16 h and analyzed by
flow cytometry with FITC-labeled annexin V and propidium iodide. The
fluorescence of cells less intensely stained with propidium iodide
(bottom area in the left panels) was replotted to
show the binding of annexin V (right panels).
Numbers in the left panels indicate the
percentages of cells in the corresponding areas. Solid and
broken vertical lines in the right panels
indicate the peak fluorescence level of mock- and wild-type
virus-infected cells, respectively. The data are from one experiment of
at least three with similar results.
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Phagocytosis of Cells Infected with Mutant Virus by
Macrophages--
We then conducted phagocytosis assays with HeLa cells
that had been infected with either the wild-type or the mutant
influenza virus and cultured at a non-permissive temperature for
16 h (panels A and B in Fig.
3). The two cell populations used as
targets of macrophages differed in the activity of NA but were at
almost the same stage of apoptosis in terms of plasma membrane
integrity, chromatin condensation, and PS externalization (see Figs.
1A and 2). HeLa cells became susceptible to phagocytosis by
mouse peritoneal macrophages when infected with the wild-type virus, as
reported previously (26). However, cells infected with the mutant virus were phagocytosed with reduced efficiency compared with cells infected
with the wild-type virus. This indicates that virus-infected cells with
reduced levels of NA activity were less efficiently phagocytosed by
macrophages than those with wild-type NA. We then compared the PS
dependence of the phagocytosis reactions using cells infected with the
two virus strains. The phagocytosis of both cell populations was
severely and specifically inhibited by the addition of PS-containing
liposomes (panel C in Fig. 3), indicating that influenza
virus-infected cells are phagocytosed by macrophages in a PS-mediated
manner irrespective of the activity of NA.
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Fig. 3.
Phagocytosis of influenza virus-infected
cells by macrophages. HeLa cells infected with either the
wild-type or the mutant influenza virus together with mock-infected
cells were cultured at a non-permissive temperature (40 °C) for
16 h and subjected to phagocytosis assays with mouse peritoneal
macrophages. A, light micrographs of macrophages after
phagocytosis reactions. The engulfed HeLa cells are shown by
white-appearing fluorescence. Bar = 50 µm.
B, quantification of phagocytosis. *, significantly
different, p < 0.001. C, effect of
liposomes. Phagocytosis reactions were conducted in the presence of PS
or PC liposomes (1 mM). The extent of phagocytosis is shown
relative to that in the control reaction with no added liposomes, which
was taken as 100. The means of the phagocytic index in the reactions
with no liposomes were 18 ± 2.5 and 13 ± 1.5 for the cells
infected with the wild-type and the mutant virus, respectively. *,
significantly different, p < 0.001.
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Phagocytosis of Virus-infected Cells Cultured in the Presence of
Zanamivir--
The above results indicate that the activity of NA in
influenza virus-infected cells is needed for the efficient phagocytosis of those cells by macrophages. To further confirm this, we examined the
effect of zanamivir, a sialic acid derivative that specifically binds
to and inhibits NA (32). HeLa cells were infected with the wild-type
influenza virus and cultured in the presence or absence of zanamivir at
a concentration that inhibits the NA activity (32). Treatment with the
inhibitor did not alter the growth of virus, as indicated by the
following observations. The proportions of cells positive for
immunofluorescence with the anti-influenza virus antiserum were 100 and
99 ± 1.4% for cells treated and not treated with zanamivir,
respectively (Fig. 4A). We
found no difference in the production of viral proteins in the two cell
populations by Western blotting (Fig. 4B). In addition, both
cell populations appeared to be similarly apoptotic; there was no
significant difference in the extent of plasma membrane integrity or
chromatin condensation (data not shown). However, PS externalization
seemed to be enhanced in the presence of zanamivir (Fig.
4C). This, together with the result shown in Fig.
2C, suggested that a defect in the NA activity leads to
stimulation of PS externalization in influenza virus-infected cells.
These results do not agree with those reported by other investigators who showed that apoptosis was inhibited by zanamivir (22). The reasons for this discrepancy are not clear at present but
could be due to the difference in the host cell strains used in the two
studies; influenza virus propagates more actively in Madin-Darby
canine kidney cells used by Morris et al. (22) than in HeLa cells.
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Fig. 4.
Virus growth and apoptosis induction in the
presence of zanamivir. HeLa cells that had been infected with the
wild-type influenza virus and cultured at 37 °C for 20 h in the
presence or absence of 10 µM zanamivir were analyzed for
the growth of virus by immunofluorescence (A) or Western
blotting (B) using the anti-influenza virus antiserum and
for PS externalization (C). Bar = 50 µm.
The symbols in B and C are the same as those used
in Figs. 1 and 2. The data are from one experiment of at least three
with similar results.
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When the cells were subjected to the phagocytosis assay, macrophages
engulfed zanamivir-treated cells to a lesser extent than untreated
cells (Fig. 5). The inhibitory effect was
not observed when the drug was present only during the phagocytosis
reaction (data not shown). Treatment of cells with another sialic acid derivative,
2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid
(Sigma), a weaker NA inhibitor than zanamivir (32), caused a similar effect but only at a higher concentration (20 mM) (data not
shown). These results indicate that NA needs to be active for influenza virus-infected cells to be efficiently phagocytosed by macrophages.
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Fig. 5.
Phagocytosis of influenza
virus-infected cells cultured in the presence of zanamivir. HeLa
cells infected with the wild-type influenza virus were cultured at
37 °C for 20 h in the presence or absence of 10 µM zanamivir and subjected to the phagocytosis assay.
A, light micrographs of macrophages after phagocytosis
reactions. Bar = 100 µm. B, quantification
of phagocytosis. *, significantly different, p < 0.002.
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Binding of Sialic Acid-recognizing Lectins to Influenza
Virus-infected Cells--
All of the above results made us anticipate
that NA modifies the structure of sugar chains at the surface of
influenza virus-infected cells and that the resulting sugar moieties
participate in the efficient phagocytosis of infected cells by
macrophages. To verify this possibility, changes in the amount of
sialic acid on the surface of HeLa cells before and after influenza
virus infection were analyzed. For this purpose, the binding of sialic
acid-recognizing lectins to the surface of HeLa cells was assessed by
flow cytometry (Fig. 6). The results
clearly showed that the binding of three different lectins that
recognize (though not exclusively) sialic acid was reduced upon
infection with the wild-type virus (panel A). The decrease
in the efficiency of lectin binding was almost completely cancelled
when cells were infected with the mutant virus (panel A) or
significantly weakened when cells were infected with the wild-type
virus and cultured in the presence of zanamivir (panel B).
These results indicate that the amount of cell surface sialic acid
decreases upon infection with influenza virus in a manner dependent on
the activity of NA. We then determined a time course of this event.
Cells infected with the wild-type virus were collected at various time
points and analyzed for the binding of wheat germ agglutinin in
comparison with that of annexin V (panel C). The decrease in
the efficiency of lectin binding was first detectable as early as
3 h after the culturing started, and the level of lectin binding
reached a plateau by 6 h. Essentially the same results were
obtained using the other two lectins (data not shown). This change
occurred much earlier than the externalization of PS, which became
obvious at 9 h being maximized by 16 h. Because phagocytosis
of virus-infected cells by macrophages reaches significant levels at
9 h (27), surface desialylation by itself seems insufficient for
phagocytosis induction.
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Fig. 6.
Binding of sialic acid-recognizing lectins to
influenza virus-infected cells. Virus-infected HeLa cells were
mixed with FITC-labeled lectins and propidium iodide and analyzed by
flow cytometry. Binding of lectins to cells less intensely stained by
propidium iodide is shown. Broken vertical lines indicate
the peak fluorescence level of mock-infected cells (A and
B) or uninfected cells (C). The results of
control cells with no added lectins are marked with none.
The lectins used were: WGA, wheat germ agglutinin;
MAM, M. amurensis lectin; SSA,
S. sieboldiana lectin. The data are from one experiment of
at least three with similar results. A, HeLa cells infected
with either the wild-type or the NA mutant virus (bottom
panels) together with control mock-infected cells (top
panels) were cultured at a non-permissive temperature (40 °C)
for 16 h and analyzed. B, wild-type virus-infected HeLa
cells were cultured at 37 °C for 20 h in the presence or
absence of zanamivir and analyzed (bottom panels). Top
panels show the results with control mock-infected cells.
C, HeLa cells infected with the wild-type virus for the
indicated periods were analyzed for the binding of wheat germ
agglutinin or annexin V.
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We finally examined the effect of exogenously added sugars on
phagocytosis. Phagocytosis assays with cells infected with the wild-type virus for 20 h were conducted in the presence of Neu5Ac (0.01~10 mM), GlcNAc (20 or 50 mM), Gal (40 mM), or Man (40 mM), but they neither
stimulated nor inhibited the reaction (data not shown). This suggests
that macrophages recognize not monosaccharides but more complex sugar
moieties that are exposed at the surface of influenza virus-infected
cells as a result of desialylation.
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DISCUSSION |
Macrophages specifically recognize "phagocytosis markers"
present on the surface of apoptotic cells using corresponding receptors (4, 5). Although a number of molecules have been proposed as candidate
phagocytosis markers, it is unclear whether each marker functions
independently or whether more than two molecules are simultaneously
recognized by macrophages. PS, the best characterized phagocytosis
marker, seems to be capable of inducing phagocytosis by itself in
macrophages, because its surface expression independent of apoptosis
makes cells susceptible to phagocytosis (34, 41). On the other hand, it
is also possible that PS cooperates with other phagocytosis markers to
make apoptotic cells more effectively recognized by macrophages
(12-14). Fadok, Henson, and co-workers recently showed that ligation
between candidate phagocytosis markers other than PS and corresponding
receptors led to the binding of macrophages to apoptotic cells, but
engulfment did not take place unless the PS receptor co-existed on the
surface of macrophages (15). They have proposed the "tether
and tickle" mechanism for heterophagic clearance of apoptotic cells;
that is, two distinct receptors, which are responsible for the binding
of phagocytic cells to apoptotic cells and for transmission of the
signal to induce engulfment, are required (44).
In the present study, we showed that influenza virus-infected cells
need to have both externalized PS and functional NA to be maximally
recognized and phagocytosed by macrophages. PS most likely serves as a
phagocytosis-inducing ligand that is probably recognized by a signaling
receptor, i.e. the macrophage PS receptor (42). As for the
role of NA, it is likely that sialic acids located at the ends of sugar
chains are cleaved off by NA, and the resulting asialoglycomoieties
then act as tethering ligands. In fact, changes of the glycan structure
by desialylation occur during apoptosis, and cells undergoing apoptosis
are bound by the asialoglycoprotein receptor present on the surface of
Kupffer cells (phagocytes in the liver) (45, 46). Our previous study showed that macrophage phagocytosis of influenza virus-infected cells
was almost completely inhibited by the addition of PS liposomes at all
times during the culture period (27). In addition, we found in the
present study that PS liposomes inhibited the phagocytosis of cells
infected with the wild-type and the NA mutant virus to very similar
extents and that loss of cell surface sialic acids by itself did not
seem to induce phagocytosis. These results collectively suggest that
the surface change(s) caused by NA act in cooperation with PS for
efficient recognition and engulfment of virus-infected cells by macrophages.
The extent of phagocytosis by macrophages continues to increase even
after desialylation and PS externalization at the surface of
influenza virus-infected cells are maximized (27). Other molecules
should thus participate in macrophage recognition of virus-infected
cells. Scott et al. (47) showed that a monoclonal antibody,
which recognizes another viral envelope protein, HA, inhibits the
binding of human monocytes to Madin-Darby canine kidney cells
infected with influenza A/WSN (H1N1) virus. A group of molecules
bound by the collagenous C-type lectin called collectin are also likely
candidates; phagocytosis of apoptotic neutrophils by alveolar
macrophages was specifically stimulated by the lung-specific collectins, surfactant proteins A and D (48). It can thus reasonably be
speculated that many kinds of molecules cooperate in the recognition of
influenza virus-infected cells by macrophages so that those cells are
effectively removed by phagocytosis before the infection spreads in the body.
Influenza virus-infected cells are also phagocytosed by dendritic cells
in an apoptosis-dependent manner, and this phagocytosis appears to lead to antigen presentation and activation of CD8-positive T lymphocytes (43, 49). We previously showed that phagocytosis of
influenza virus-infected cells by macrophages results in the inhibition
of virus growth (21). It is therefore likely that apoptosis-dependent phagocytosis of influenza
virus-infected cells may protect the organism from viral invasion in
two different ways. Further experiments are needed to examine whether
such self-defense mechanisms against viral diseases really function
in vivo.
| |
ACKNOWLEDGEMENTS |
We thank GlaxoSmithKline for the gift of
zanamivir, E. Nobusawa for suggestions on the NA assay, Y. Suzuki for
suggestions on the use of NA inhibitors, and C. Fujii for help with
macrophage preparation and flow cytometry.
| |
FOOTNOTES |
*
This work was supported by a grant-in-aid for scientific
research from the Japan Society for the Promotion of Science and a
grant from the Sumitomo Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-76-234-4481; Fax: 81-76-234-4480; E-mail:
nakanaka@kenroku.kanazawa-u.ac.jp.
Published, JBC Papers in Press, March 7, 2002, DOI 10.1074/jbc.M201074200
| |
ABBREVIATIONS |
The abbreviations used are:
PS, phosphatidylserine;
PC, phosphatidylcholine;
FITC, fluorescein
isothiocyanate;
HA, influenza virus hemagglutinin;
NA, influenza virus
neuraminidase;
NP, influenza virus nucleoprotein;
PBS, phosphate-buffered saline;
ts, temperature-sensitive.
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