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Journal of Virology, July 2001, p. 5921-5929, Vol. 75, No. 13
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.73.13.5921-5929.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Human Lymphocyte Apoptosis after Exposure to
Influenza A Virus
Joan E.
Nichols,1,*
Jean A.
Niles,1 and
Norbert J.
Roberts Jr.1,2
Division of Infectious Diseases, Department
of Internal Medicine,1 and Department of
Microbiology and Immunology,2 University of
Texas Medical Branch, Galveston, Texas 77555-0435
Received 27 December 2000/Accepted 9 April 2001
 |
ABSTRACT |
Infection of humans with influenza A virus (IAV) results in a
severe transient leukopenia. The goal of these studies was to analyze
possible mechanisms behind this IAV-induced leukopenia with emphasis on
the potential induction of apoptosis of lymphocytes by the virus.
Analysis of lymphocyte subpopulations after exposure to IAV showed that
a portion of CD3+, CD4+, CD8+, and
CD19+ lymphocytes became apoptotic (terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
positive). The percentage of cells that are infected was shown to be
less than the percentage of apoptotic cells, suggesting that direct
effects of cell infection by the virus cannot account fully for the
high level of cell death. Removal of monocytes-macrophages after IAV
exposure reduced the percent of lymphocytes that were apoptotic.
Treatment of virus-exposed cultures with anti-tumor necrosis factor
alpha did not reduce the percentage of lymphocytes that were apoptotic.
In virus-exposed cultures treated with anti-FasL antibody, recombinant
soluble human Fas, Ac-DEVD-CHO (caspase-3 inhibitor), or Z-VAD-FMK
(general caspase inhibitor), apoptosis and production of the active
form of caspase-3 was reduced. The apoptotic cells were
Fas-high-density cells while the nonapoptotic cells expressed a low
density of Fas. The present studies showed that Fas-FasL signaling
plays a major role in the induction of apoptosis in lymphocytes after exposure to IAV. Since the host response to influenza virus commonly results in recovery from the infection, with residual disease uncommon,
lymphocyte apoptosis likely represents a part of an overall beneficial
immune response but could be a possible mechanism of disease pathogenesis.
| |
INTRODUCTION |
Influenza virus has been shown to
induce apoptosis in tissue culture cells (18, 43) and in
peripheral blood monocytes (14, 19). A depletion of
lymphocytes due to apoptosis has also been described in mice infected
with a highly virulent influenza A virus (IAV) (H5N1) isolated from
humans (46). The immunopathological mechanisms and the
role played by the virus infection of leukocytes with respect to
disease pathology in general and leukocyte death in particular have not
been elucidated. An early lymphopenia has been described in
IAV-infected patients (7, 10, 24), and inoculation of
humans with IAV has been shown to cause a decrease in both T- and
B-cell numbers during illness (7, 10). In the experimental
infections, volunteers developed a severe T-cell lymphopenia and a
moderate B-cell lymphopenia even though seroconversion occurred in 90%
of the volunteers, suggesting that T- and B-cell functions were
preserved (10, 12). This observed lymphopenia could be the
result of cell migration from the circulation and/or cell death caused
by necrosis or by apoptosis or through suppression of hematopoeisis.
Fas and FasL have been shown to play a role in the induction of
apoptosis of activated mature T cells at the culmination of an immune
response (21, 32) and in the killing of virus-infected or
neoplastic cells by cytotoxic T cells (48). One of the
best-characterized death receptors, Fas (CD95) is a 48-kDa
transmembrane glycoprotein belonging to the tumor necrosis factor (TNF)
receptor family (29, 31, 32). Fas has been shown to be
involved in the induction of apoptosis when cross-linked with anti-Fas
antibodies (21, 49) or Fas ligand (FasL)
(42). FasL is a 40-kDa TNF family member protein that
induces apoptosis by binding to Fas, its cell surface receptor. FasL
expression on cytotoxic T cells can induce cytolysis of target cells
expressing Fas (26, 42). Resting monocytes-macrophages
express a low level of Fas receptor but no FasL. Once activated, these
cells express increased Fas as well as FasL, which is rapidly expressed
after mobilization from presynthesized stores (26). It has
been suggested that monocytes-macrophages can trigger apoptosis in
other types of cells by regulated expression of FasL on their cell
surface and by release of soluble FasL (5).
Apoptosis signal transduction and induction is associated with the
coordinated action of a series of caspases (aspartate-specific cystein
proteases) (13, 23, 40, 45). Following binding of Fas to
FasL, trimerization of Fas recruits the Fas-associated death domain
(FADD) through interactions of Fas and FADD. This step is followed by
caspase-8 binding, and interactions between FADD and caspase-8 result
in the activation of caspase-8. Activation of caspase-8 initiates the
activation of a cascade of caspases including caspase-3 (22, 23,
28). Caspase-3 activities have been shown to control both the
cytoplasmic and nuclear events associated with Fas-mediated apoptosis
(51).
In this study we analyzed apoptosis and expression of Fas (CD95), FasL,
and the active form of caspase-3 by peripheral blood mononuclear
leukocytes (MNL) that were exposed to IAV. We determined that apoptosis
does occur in cells exposed to IAV, and we present data suggesting a
role for Fas-FasL-mediated induction of apoptosis in peripheral blood lymphocytes.
| |
MATERIALS AND METHODS |
Virus stocks.
Influenza A/AA/Marton/43 (H1N1) virus was
grown in allantoic cavities of 10-day-old embryonated hen's eggs. The
allantoic fluid was pooled after collection and frozen at 
70°C
until titered to 107 or 108 when assayed in
Madin-Darby canine kidney (MDCK) cells (American Type Culture
Collection, Rockville, Md.) or used for exposure of MNL
(37). For sham exposures, allantoic fluid from uninfected eggs was collected, pooled, and frozen at 70°C until used.
Influenza A virus strains A/Bethesda/85 (H3N2) (wild type, termed wt
A/Bethesda) and A/Ann Arbor/6/60 × A/Bethesda/85 (cold adapted, termed
ca A/Bethesda) were a kind gift of Brian Murphy (National Institute of
Allergy and Infectious Diseases, Bethesda, Md.). Heat-inactivated A/AA/Marton/43 was prepared by incubation for 1 h at 56°C.
Collection of MNL and exposure to virus.
Informed consent
for withdrawal of blood was obtained from all donors. Donors ranged in
age from 18 to 45 years. Equal numbers of male and female subjects were
used as volunteer donors. It was expected that all donors had
experienced past in vivo exposure to IAV.
MNL were obtained from the peripheral blood of healthy human volunteers
by Ficoll-Hypaque sedimentation (Pharmacia) (3). Cells
were counted and viability was determined (i) by the ability to exclude
trypan blue under light microscopy or (ii) by the use of propidium
iodide with analysis by fluorescent microscopy and flow cytometry.
MNL were exposed or sham exposed to influenza virus at a multiplicity
of infection of 1 for 1 h at 37°C in serum-free RPMI
1640 supplemented with 2 mM glutamine, 100 U of penicillin G,
and 100 µg
of streptomycin/ml (
37). For sham exposures, cells
were
exposed to a volume of chicken allantoic fluid equal to that
used for
virus infections. After 1 h of exposure or sham exposure
to virus,
the MNL or purified subpopulations of cells were washed
in warm medium,
centrifuged, and reincubated at 37°C in medium
supplemented with 10%
heat-inactivated defined fetal calf serum
(Hyclone).
Monocytes-macrophages were depleted from cultures by adherence after
1 h of serum-free cell culture followed by staining of
residual
monocytes-macrophages with anti-CD14 antibody and gating
for cell
sorting on CD14-negative cells. The sorted cells were
collected,
resuspended in warm culture medium, and reincubated
at 37°C in medium
supplemented with 10% heat-inactivated defined
fetal calf serum
(Hyclone).
Flow cytometry analyses.
Acquisition, cell sorting, and
analysis were done using a FACSort (Becton Dickinson). For analyses of
lymphocytes or monocytes-macrophages, forward versus side light scatter
was used for gating. Sorting based on cell phenotype was performed by
gating on the fluorescent population to be collected, or for
monocyte-macrophage depletion experiments, gates for cell collection
were set after CD14 staining (with gating on unstained,
non-CD14+ cells). Acquisition and analysis were performed
using Cellquest software (Becton Dickinson). Calibration of the
equipment for validation of the logarithmic linearity required for the
estimation of the number of molecules of CD95 was accomplished by using
Spherotech Rainbow particles (Spherotech). Quantitation of surface
molecule expression was done using Quanticalc Software (Becton Dickinson).
Staining for analyses of cell phenotype.
Phenotypes of cells
were determined by using monoclonal antibodies (MAbs) to identify
monocytes-macrophages (CD14+) or CD3+,
CD4+, CD8+, and CD19+ lymphocyte
subsets (Becton Dickinson). Antibodies were conjugated to fluorescein
isothiocyanate (FITC), phycoerythrin (PE), or peridinin chlorophyll
protein (PerCP), and corresponding immunoglobulin G (IgG)-matched
isotype control antibodies were used to set baseline values for
analysis markers. For surface staining, appropriate concentrations of a
single MAb, combinations of multiple MAbs, or IgG-matched control
antibodies were mixed with cells and treated as described by the
manufacturer (Becton Dickinson or BD Pharmingen). After fixation in 2%
paraformaldehyde (PAF), cells were stored at 4°C until fluorescent
microscopy and/or flow cytometric analysis was performed.
Analyses of cell apoptosis.
Apoptosis was determined by
quantitation of DNA strand breaks using the terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
(TUNEL) assay (In Situ Cell Death kit; Boehringer Mannheim). The cell
phenotype or activation state was established by cell surface labeling
with MAbs prior to the TUNEL reaction. After being stained, cells were
fixed in 2% PAF and permeabilized for 10 min using 0.6%
n-octyl glucopyranoside (Sigma Chemical Co.). The TUNEL
reaction was carried out as described in the manufacturer's instructions. In a subset of experiments, the apoptosis was confirmed after cell sorting by demonstration of DNA fragmentation resulting in
the presence of bands of 180 bp or multiples thereof, forming a
characteristic ladder effect on gel electrophoresis (15).
Analyses of viral protein production.
Cells were stained for
surface expression of CD3, CD4, or CD8 prior to staining for viral
protein expression. After fixation in 2% PAF, the cells were
permeabilized for 10 min in 0.6% n-octyl glucopyranoside
(Sigma Chemical Co.). Expression of influenza virus proteins was
determined by indirect immunofluorescent staining using goat anti-H1
hemagglutinin, anti-N1 neuraminidase, and anti-M polyclonal antisera
(National Institutes of Health [NIH] reference reagents). Cells were
incubated in 20 µl of a 1:500 dilution of goat anti-influenza virus
antibodies for 1 h. After this step cells were washed with
Dulbecco's phosphate-buffered salt solution (DPBS) and stained with 20 µl of a 1:500 dilution of FITC-conjugated rat anti-goat IgG antibody
(Organon Teknika) for 1 h.
Analyses of Fas, FasL, and active caspase-3 expression.
The
expression of membrane-bound Fas (CD95) and FasL by lymphocytes and
monocytes was measured by flow cytometry of sham-exposed and
virus-exposed MNL. Cells were then stained for Fas or FasL expression.
Antibody-binding capacity can be used to determine values of antigen
density on the cell surface, since the binding capacity can be related
directly to the level of antigen expression on the surface of a cell
(9). Quantitative analyses using this assay were performed
to evaluate the expression of CD95 by CD3+,
CD4+, and CD8+ lymphocytes. For the
quantitation estimates of cell surface molecules, 106 MNL
were incubated with 20 µl of either anti-CD3, -CD4, or -CD8 conjugated to PerCP (BD Pharmingen) and 20 µl of anti-CD95 conjugated to PE (Becton Dickinson). Antibody labeling was performed for 0.5 h at
4°C. Cells prepared for quantitations of surface molecules on
CD4+ and CD8+ lymphocytes were analyzed
immediately after staining without fixation of cells. Cells prepared
for analysis of CD95 expression (percent positive) and apoptosis were
fixed in 2% PAF. The percent apoptotic cells for CD3+
CD95+ lymphocytes was determined by using the TUNEL assay.
Quantitation of immunofluorescence intensity was used to estimate the
average number of molecules expressed on cells (
8,
16).
Calibration of the equipment was done using Spherotech
Beads
(
20). Fluorescence quantitation was done using the
Quantiquest
system (Becton Dickinson). Samples were gated on
lymphocytes by
using forward and side scatter gates, and then cursors
were set
to measure the median relative fluorescence intensity (RFI) of
the cell population under study (
20). For the median RFI
on
CD3
+, CD4
+, or CD8
+ T-cell
measurements, a light scatter gate was combined with a
second gate set
on cells positive for CD3, CD4, or CD8 and CD95.
Expression of CD95
molecules on CD3
+, CD4
+, or CD8
+
cells was measured by gating on both dim and brightly staining
cells.
For FasL staining, 20 µl of a 1:100 dilution of anti-FasL antibody
(clone NOK-1; BD Pharmingen) was added to appropriate samples.
After a
0.5-h incubation, cells were washed with DPBS and stained
with 20 µl of a 1:500 dilution of FITC-conjugated goat anti-mouse
F(ab)
2 antibody (Organon Teknika) for 0.5 h. Cells
were analyzed
by fluorescent microscopy and flow cytometry without
fixation.
In a set of experiments after 1 h of sham exposure or exposure to
IAV, 1 µg of soluble recombinant human Fas/ml (
6) or
1 µg of azide-free neutralizing anti-FasL antibody (clone NOK-1)/ml
was
added to cultures of MNL to determine the influence of Fas-FasL
signaling on production of the active form of caspase-3 or the
induction of apoptosis after IAV exposure. Purified isotype-matched
control antibodies (mouse IgG1, clone MOPC-21) were added to sham-
and
virus-exposed cultures and had no effect on caspase-3 production
or
apoptosis induction. To assess the role of Fas-FasL-triggered
caspase
activation in IAV exposure-induced apoptosis, each of
the inhibitors
Z-VAD-FMK (general caspase inhibitor) (20 µM),
AC-DEVD-CHO (caspase-3
inhibitor) (20 µM), and the control inhibitor
Z-FA-FMK (20 µM) was
added to a set of MNL cultures (
17,
39).
The caspase-3
level was evaluated by staining 10
6 sham-exposed or
virus-exposed MNL with 20 µl of PerCP-conjugated
anti-CD3 antibody
(
44). After 30 min the cells were washed and
fixed in 2%
PAF for 2 h. The CD3
+-stained cells were permeabilized
for 10 min using 0.6%
n-octyl
glucopyranoside (Sigma
Chemical Co.) and then stained for caspase-3
expression with 20 µl of
FITC-conjugated rabbit anti-active caspase-3
MAb (clone C-92-605) (BD
Pharmingen). After 30 min the cells were
washed and analyzed
immediately. The percent apoptotic cells for
all cell treatments was
determined by using the TUNEL
assay.
In another subset of experiments, after 1 h of sham exposure or
exposure to IAV, 1 µg each of stimulatory anti-human Fas antibody
(clone DX2), agonistic anti-FasL antibody (clone G247-4), anti-TNF-
antibody (clone Mab1), or anti-ICAM-1 (clone 84H10) (AMAC Inc.)
antibody/ml was added to cultures of MNL to determine the influence
of
Fas-FasL, TNF-
, or cell clustering and cell-cell interactions,
respectively, on induction of apoptosis in CD4
+ or
CD8
+ lymphocytes. Emetine (10
5 M) (Sigma
Chemical Co.) was also added to a set of MNL cultures
3 h (to allow
synthesis of viral proteins) after sham exposure
or exposure to IAV.
Cells were harvested after 24 h, washed, stained
with anti-CD4 or
-CD8 PerCP-conjugated antibodies, and then fixed
with 2% PAF. The
percent apoptotic cells for these cell treatments
was determined using
the TUNEL
assay.
Statistical analyses.
Results are expressed as the mean ± the standard deviation (SD) for the stated number of experiments. The
statistical significance of differences in the means was calculated
using Student's t test performed by EXCEL software
(Microsoft, Inc., Bothwell, Wash.). Mean differences in the values were
considered significant when P was less than 0.05.
| |
RESULTS |
Both monocytes-macrophages (47% ± 6%) and lymphocytes (7% ± 2%), identified by forward and side light scatter properties, expressed viral proteins 24 h after exposure. The percentage of monocytes-macrophages positive for the production of viral proteins was
slightly higher at 48 and 72 h after exposure but there was no
significant increase in the percentage of total lymphocytes positive
for the production of viral proteins at the later time points. Of the
CD3+ T cells, 4.6% ± 0.87% at 24 h and 5.1% ± 0.4% at 48 h were positive for the production of viral proteins.
Among the lymphocyte subsets, CD3+, CD4+,
CD8+, and CD19+ cells were all shown to produce
viral proteins after IAV exposure (data not shown).
Lymphocyte apoptosis.
To detect apoptosis in lymphocytes,
CD3+, CD4+, CD8+, and
CD19+ cells were sorted (99 to 100% pure for the labeled
cell type) and DNA fragmentation was assayed using agarose gel
electrophoresis of DNA extracted from cells 24 h after sham
exposure or virus exposure. Characteristic DNA ladders were seen only
in CD3+, CD4+, CD8+, and
CD19+ virus-exposed cells and never in sorted cells from
sham-exposed cultures (data not shown). The TUNEL assay was then used
to detect DNA strand breaks indicative of apoptosis on a single-cell basis.
Measurable levels of apoptosis were seen in CD3
+ T cells
after exposure to virus but not in sham-exposed cells harvested at
24 h (
P = 0.03), 48 h (
P = 0.0016), or 72 h (
P = 0.009). The percentage
of CD3
+ cells that were apoptotic was reduced in
virus-exposed, monocyte-macrophage-depleted
cultures at 24 h
(
P = 0.069) and was significantly reduced at
48 (
P = 0.017) and 72 h (
P = 0.004) (Fig.
1). If monocytes-macrophages
were removed
1 h after exposure to virus and then added back 24
h later,
levels of apoptosis seen in CD3
+ cells increased from
levels that were seen for depleted cultures
but did not reach the level
seen for undepleted MNL cultures (data
not shown).
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FIG. 1.
Role of monocytes-macrophages in induction of
CD3+ lymphocyte apoptosis. MNL were sham exposed or exposed
to virus, and after 1 h cultures were sham depleted (solid bars)
or depleted (hatched bars) of monocytes-macrophages. Results from five
experiments are presented as the mean percentage of apoptotic
(TUNEL+) CD3+ cells ± SD. Data from
10,000 CD3+ cells were collected for each sample.
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Analysis of lymphocyte subsets showed that apoptosis was seen in
CD3
+, CD4
+, CD8
+ (Fig.
2), and
CD19
+ (data not shown) cells. Sham-exposed
CD3
+, CD4
+, CD8
+, and
CD19
+ cells exhibited levels of staining similar to that
seen for the
TUNEL-negative assay controls (less than 1% positive).
Significant
increases in the percentage of apoptotic cells in
virus-exposed
compared to sham-exposed cell cultures were seen at
24 h for CD3
+ (
P = 3.69 × 10
5), CD4
+ (
P = 0.00053), and CD8
+ (
P = 1.85 × 10
5) cells and at 48 h for CD3
+
(
P = 0.003), CD4
+ (
P = 0.022), and CD8
+ (
P = 0.001) T-cell
subpopulations. A significantly higher percentage
of CD8
+
cells was seen to be TUNEL positive after exposure to virus than
similarly treated CD4
+ cells (
P = 0.009 and
P = 0.030 at 24 and 48 h, respectively)
(Fig.
2).
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FIG. 2.
Apoptosis-related cell death of sham-exposed and
virus-exposed CD3+ (solid bars), CD4+ (open
bars), and CD8+ (hatched bars) lymphocytes. Results from
five experiments are presented as mean percentage of apoptotic
(TUNEL+) cells ± SD. Data from 10,000 CD3+, CD4+, or CD8+ cells were
collected for each sample.
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Measurement and quantitation of CD95.
Surface Fas
(CD95) expression was examined for gated populations of
CD4+ cells and CD8+ cells. There was no
statistically significant increase in expression of CD95 by
virus-exposed CD4+ cells compared to sham-exposed cells at
any of the time points measured (Fig.
3A). The percentage of virus-exposed
CD8+ cells expressing CD95 increased rapidly after exposure
to virus (Fig. 3B) and was significantly different from that of
sham-exposed cells at 24 h (P = 0.022), 48 h
(P = 0.001), 72 h (P = 0.037), and
96 h (P = 0.047). The average number of molecules
of CD95 per cell was quantitated before and after exposure to IAV.
There was a significant decrease in expression of CD95 by virus-exposed CD4+ cells at 12 h (P = 0.046) but
levels were increased on virus-exposed compared to sham-exposed
CD4+ cells at 24 h (P = 0.041) and 48 h (P = 0.033) (Fig. 3C). The expression of CD95
increased significantly on virus-exposed compared to sham-exposed
CD8+ cells at 12 h (P = 0.048) and
24 h (P = 0.001) (Fig. 3D). The differences in
expression of CD95 by virus-exposed CD4+ versus
CD8+ cells at 12 h (P = 0.038) and
24 h (P = 0.00046) were significant.
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FIG. 3.
CD95 expression by lymphocytes after sham exposure (open
symbols and bars) or exposure to virus (solid symbols and bars).
Results from five experiments are presented as the mean percentage of
CD4+ (A) or CD8+ (B) cells that were CD95
positive ± SD and as the mean number of molecules of CD95
expressed on the surface of the CD4+ (C) and
CD8+ (D) cells. For each time point, the data from 10,000 CD4+ or CD8+ cells were collected.
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The average number of molecules (density) of CD95 on virus-exposed
CD3
+ TUNEL-positive and CD3
+ TUNEL-negative
lymphocytes was determined. The density of CD95
on CD3
+
TUNEL-positive compared with CD3
+ TUNEL-negative
lymphocytes was increased significantly at 24
h (
P = 0.00039) (Fig.
4).
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FIG. 4.
Level of CD95 density (average number of molecules per
cell) for TUNEL-negative (open bar) and TUNEL-positive (solid bar)
CD3+ lymphocytes 24 h after exposure to IAV. The data
from 20,000 CD3+ cells were collected.
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FasL expression by virus-exposed cells and influence of
anti-Fas and anti-FasL antibodies.
FasL expression by
virus-exposed MNL was increased compared to sham-exposed MNL (Fig.
5). The majority of FasL was expressed on
monocytes-macrophages within the MNL, but small increases were noted in
the lymphocyte population as well (gating was done on CD3+
cells; data not shown) (P = 0.0005) at 24 h.
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FIG. 5.
(A) Data from one representative experiment showing FasL
expression in IAV-exposed MNL (solid histogram) and sham-exposed MNL
(grey line). For each sample, data from 10,000 cells were collected.
(B) FasL expression by MNL after sham exposure (open bar) or exposure
to virus (solid bar). Results from five experiments are presented as
the mean percentage of cells that expressed FasL ± SD. For each
sample, data from 10,000 cells were collected.
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The production of active caspase-3 was measured in CD3
+
lymphocytes (Fig.
6A and B).
Significantly more caspase-3 was produced
by IAV-exposed MNL cultures
(
P = 3.9 × 10
5) than sham-exposed
cultures (Fig.
6B). In IAV-exposed MNL the
levels of caspase-3
production were significantly decreased in
cultures treated with
anti-FasL antibody (
P = 0.002) and soluble
recombinant
human Fas (
P = 0.001), as well as with the caspase
inhibitors Z-VAD-FMK (
P = 2.3 × 10
5) and AC-DEVD-CHO (
P = 1.48 × 10
5). Sham-exposed cultures exhibited minimal levels
of caspase-3
production (<1.4%), and treatment of these cultures with
anti-FasL
antibody, soluble recombinant human Fas, or the caspase
inhibitors
Z-VAD-FMK and AC-DEVD-CHO did not significantly increase or
decrease
the level of caspase-3 (Fig.
6B). Treatment of sham-exposed
cultures
with the control inhibitor Z-FA-FMK (mean, 1.05 ± 0.11)
did not
increase or decrease the level of caspase-3 production.
Treatment
of IAV-exposed cultures with the control inhibitor Z-FA-FMK
did
not result in a decrease in caspase-3 production (mean, 29.46
± 11.8).
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FIG. 6.
(A) Data from one representative experiment showing
production of the active form of caspase-3 (upper row of histograms) or
the induction of apoptosis (lower row of histograms) in IAV-exposed
CD3+ cells (solid histograms). The sham-exposed caspase-3
level or percent apoptosis is shown as the gray line in the far left
histogram of each row. The effect of treatment of IAV-exposed cells
with recombinant soluble human Fas, anti-FasL antibody, the general
caspase inhibitor Z-VAD-FMK, or the caspase-3 inhibitor AC-DEVD-CHO are
shown. Treatment of sham-exposed CD3+ cells with
recombinant soluble human Fas, anti-FasL antibody, the general caspase
inhibitor Z-VAD-FMK, or the caspase-3 inhibitor AC-DEVD-CHO produced
levels of caspase-3 less than 1.3% and levels of apoptosis less than
1%. For each sample, data from 10,000 CD3+ cells were
collected. (B) Active caspase-3 production in CD3+
sham-exposed (open bars; left graph) and virus-exposed (solid bars;
right graph) lymphocytes after treatment with anti-FasL antibody,
recombinant soluble human Fas, the general caspase inhibitor Z-VAD-FMK,
or the caspase-3 inhibitor AC-DEVD-CHO. Results represent the mean
percentage of apoptotic cells ± SD from five experiments, showing
the effect of the above treatments. For each sample, data from 10,000 CD3+ cells were collected. (C) Apoptosis of
CD3+ sham-exposed (open bars; left graph) and virus-exposed
(solid bars; right graph) lymphocytes after treatment with anti-FasL
antibody, recombinant soluble human Fas, the general caspase inhibitor
Z-VAD-FMK, or the caspase-3 inhibitor AC-DEVD-CHO. Results represent
the mean percentage of apoptotic cells ± SD from five
experiments, showing the effect of the above treatments. For each
sample, data from 10,000 CD3+ cells were collected.
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The sensitivity of CD3
+ lymphocytes from virus-exposed
cultures to Fas-FasL-induced apoptosis was evaluated through the
addition
of neutralizing anti-FasL antibody, soluble recombinant Fas,
or
the caspase inhibitors AC-DEVD-CHO and Z-VAD-FMK to sham-exposed
and
influenza virus-exposed MNL cultures. In IAV-exposed MNL,
the levels of
apoptosis were significantly decreased in cultures
treated with soluble
recombinant human anti-FasL antibody (
P =
0.0007) or
soluble recombinant human Fas (
P = 0.015), as well
as
with the caspase inhibitors Z-VAD-FMK (
P = 0.008) and
AC-DEVD-CHO
(
P = 0.017). Sham-exposed cultures
exhibited minimal levels of
apoptosis (<1.25%), and treatment of the
sham-exposed cultures
with the reagents listed above did not
significantly increase
or decrease the level of apoptosis (Fig.
6A and
C). Treatment
of sham-exposed cultures with the control inhibitor
Z-FA-FMK did
not result in an increase or decrease in the level of
apoptosis
(mean, 1.2 ± 0.45). Treatment of IAV-exposed cultures
with the
control inhibitor Z-FA-FMK did not result in a decrease in the
level of apoptosis (mean, 10.26 ± 3.2).
The level of apoptosis in CD8
+ T cells exposed to IAV was
enhanced significantly (
P = 0.027) by the addition of
anti-Fas antibodies
(Fig.
7) but
CD4
+ cells were not affected (
P = 0.20).
Blocking of FasL-Fas interactions
through the addition of anti-FasL
antibodies inhibited apoptosis
of CD8
+ cells (
P = 0.012). Addition of emetine also reduced the percentage
of
apoptotic virus-exposed CD8
+ cells (
P = 0.011) (Fig.
7). CD4
+ cells did exhibit a significant
decrease in apoptotic cells after
treatment with emetine (
P = 0.02) but not with anti-Fas (
P = 0.2)
or
anti-FasL (
P = 0.06) antibodies. Purified
isotype-matched control
antibodies were added to sham- and
virus-exposed cultures and
had no effect on caspase-3 production or
apoptosis induction.
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 7.
Apoptosis of sham-exposed and virus-exposed
CD4+ (open bars) and CD8+ (solid bars)
lymphocytes after treatment with emetine, anti-Fas antibody, anti-FasL
antibody, anti-TNF- antibody, or anti-ICAM-1 antibody. Results
represent the mean percentage of apoptotic cells ± SD from five
experiments, showing the effect of emetine (105 M), anti-Fas
antibody, and anti-FasL antibody, or from three experiments, showing
the effect of anti-TNF- antibody or anti-ICAM-1 antibody treatment
on the levels of apoptosis in CD4+ and CD8+
cells. For each sample, data from 10,000 CD4+ or
CD8+ cells were collected.
|
|
Evaluation of apoptosis in inactivated IAV and other strains of
IAV.
Significant increases in the percentage of TUNEL-positive
cells in virus-exposed compared to sham-exposed CD3+ cells
were seen after exposure to IAV strains A/AA/Marton/43, wt A/Bethesda
(P = 0.0003), and ca A/Bethesda (P = 0.0059) (Fig. 8). A significant
decrease in the percentage of TUNEL-positive cells was observed in the
inactivated virus-exposed cells compared to the infectious
virus-exposed CD3+ cells. There was also a significant
decrease in the percentage of TUNEL-positive CD3+ cells
exposed to the cold-adapted virus vaccine strain ca A/Bethesda (P = 0.024) compared to the wild-type strain wt
A/Bethesda.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 8.
Apoptosis of sham-exposed (open bar), heat-inactivated
virus-exposed (hatched bar), and virus-exposed (solid bars)
CD3+ lymphocytes. MNL were exposed to influenza A virus
strains A/AA/Marton/43 (H1N1), heat-inactivated
A/AA/Marton/43, wild-type A/Bethesda/85 (H3N2) (termed wt
A/Bethesda), or cold-adapted A/Ann Arbor/6/60 × A/Bethesda/85
(termed ca A/Bethesda) for 24 h. Results represent the mean
percentage of apoptotic cells ± SD from five experiments. For
each sample, data from 10,000 CD3+ cells were collected.
|
|
| |
DISCUSSION |
Previous studies have described a lymphopenia occurring after
natural infection with influenza A virus or administration of attenuated influenza A virus vaccine (7, 10, 12, 24). The
mechanism of this leukopenia, and specifically the role played by viral
infection of lymphocytes, has not been determined. If redistribution of
lymphocytes alone were responsible for the IAV-induced lymphopenia, an
intact functional activity of the remaining circulating lymphocytes
would be expected. Previous studies by our laboratory demonstrated that
leukocytes exposed to IAV showed proliferation in response to the virus
but concomitant depression of responses to mitogen stimulation
(37). The decreased activation that could be induced by
the presence of mitogen suggests a loss of functional activity. It is
possible that nonvirus-directed responses are suppressed in part due to
the induction of apoptosis as a result of IAV exposure. Apoptosis plays
a crucial role in the regulation of leukocyte numbers and in the
regulation of immunological response to virus infection (2, 47,
50).
Apoptosis of lymphocyte subsets.
An important step in
understanding the pathogenesis of IAV-induced leukopenia
would be to determine the role played by apoptosis in the
depletion of lymphocytes. Influenza virus has been shown to induce
apoptosis in a number of cell types, including peripheral blood
monocytes-macrophages (18), and in avian cell lines
(43). In the present studies, lymphocyte apoptosis was
detected as early as 1 day after exposure to virus. Analysis of cell
phenotypes showed that induction of apoptosis occurred in virus-exposed
but not sham-exposed CD3+, CD4+, and
CD8+ cells. Significantly more CD8+ cells than
CD4+ cells were apoptotic at both 24 and 48 h after
exposure to virus. The percentage of CD3+,
CD4+, and CD8+ lymphocytes positive for
apoptosis was significantly reduced in virus-exposed but
monocyte-macrophage-depleted cultures. In cultures in which
monocytes-macrophages were removed 1 h after exposure to virus and
then added back 24 h later, levels of apoptosis were seen to
increase from those of depleted cultures but did not reach the levels
seen in undepleted MNL cultures. These data suggest that a major
mechanism of apoptosis in these cells must involve cell-cell
interactions between monocytes-macrophages and CD3+,
CD4+, and CD8+ lymphocytes.
Role of activation and Fas-FasL in induction of apoptosis after
exposure to virus.
The Fas-FasL system is recognized as a major
pathway for the induction of apoptosis in cells. For example, in a
process referred to as activation-induced cell death, activation of T
cells can result in apoptotic death mediated by Fas (CD95)-FasL
interactions. A possible role for Fas-mediated activation-induced cell
death in CD3+, CD4+, and CD8+
lymphocytes after exposure to influenza virus warranted consideration.
An increase in the number of Fas-expressing CD4
+ and,
especially, CD8
+ cells was observed after exposure to
virus. It may not be the
expression of Fas but the quantity of Fas
expressed that controls
the induction of apoptosis in activated cells.
The density (average
number of molecules) of Fas expression also
increased on both
CD4
+ and CD8
+ lymphocytes
after exposure of the MNL cultures. The combination
of the percent
CD8
+ cells expressing Fas plus the density of Fas
expression by those
cells was most clearly evident. In a comparison
between the average
number of Fas molecules expressed on
CD8
+ and on CD4
+ cells, significant differences
were noted at 12 h (
P = 0.0038)
and 24 h
(
P = 0.00046) after exposure to influenza virus. The
increase in the density of Fas on CD3
+ cells did correlate
with the increase in apoptosis in those cells.
The highest density of
Fas was seen on CD3
+ TUNEL-positive cells (mean,
39,487 ± 6,564 molecules;
P = 0.00039)
compared
to CD3
+ TUNEL-negative cells (mean, 4,838 ± 2,227).
The expression of FasL on cells is strictly regulated and thought to be
the triggering event in the induction of Fas-FasL-mediated
apoptosis.
Monocytes-macrophages (CD14
+ cells) were the major cell
type expressing FasL but CD3
+ T cells also exhibited an
upregulation in FasL expression after
exposure to virus. Although
levels of Fas and FasL were shown
to increase after exposure to IAV,
the actual role of Fas-FasL
signaling in the induction of apoptosis was
not clear. Caspase-3
has been shown to mediate Fas-FasL induction of
apoptosis (
51).
Levels of caspase-3 increased
significantly after exposure to
IAV. Neutralizing FasL antibody,
recombinant soluble human Fas,
and the caspase inhibitors Z-VAD-FMK (a
general caspase inhibitor)
and AC-DEVD-CHO (a caspase-3-specific
inhibitor) reduced the production
of active caspase-3 and the induction
of apoptosis in IAV-exposed
CD3
+ lymphocytes. These data
support the involvement of Fas-FasL in
the induction of apoptosis in
lymphocytes after exposure to influenza
virus.
Fas-FasL-mediated killing has been shown to be sensitive to inhibition
of protein synthesis by emetine (
4,
25) although
this
treatment is fairly nonspecific. A significant decrease in
apoptotic
CD8
+ cells was seen after treatment with emetine as well as
in cells
treated with anti-FasL antibody. Although not statistically
significant,
CD4
+ cells also exhibited a decrease in
apoptosis after treatment
with emetine (
P = 0.63) or
anti-FasL antibody (
P = 0.69). In general,
however,
removal of monocytes-macrophages was more effective in
reducing the
percentage of apoptotic cells than was inhibition
of protein synthesis
using
emetine.
Induction of apoptosis by exposure to heat-inactivated virus and
other strains of IAV.
It is not clear at this time what component
of influenza virus induces apoptosis. UV-inactivated virus does not
induce apoptosis (27), nor does heat-inactivated influenza
virus (Fig. 8), although heat-inactivated IAV does activate lymphocytes
(11, 36). To understand the role that apoptosis may play
in the pathogenesis of influenza virus infection, we exposed human MNL
to wild-type as well as heat-inactivated and IAV cold-adapted vaccine
strains of influenza virus. The cold-adapted strain of virus contains the wild-type neuraminidase and hemagglutinin genes but derives all
other components of their genome from cold-adapted strains of virus.
All of the influenza infectious viruses induced significant levels of
apoptosis compared to sham-exposed cells. The wt A/Bethesda
virus
induced significantly higher levels of apoptosis than the
ca A/Bethesda
virus strain. The ca A/Bethesda strain has been
successfully used as a
vaccine strain and has not been associated
with induction of fever or
other clinical symptoms (
41). These
data suggest that the
induction of apoptosis may be associated
with strain virulence, since
this attenuated ca virus induced
less apoptosis than was seen in cells
exposed to either the A/AA/Marton/43
or wt A/Bethesda
viruses.
Future studies regarding induction of apoptosis by exposure to
virus.
Since the host response to influenza virus commonly results
in recovery from the infection, with residual disease uncommon, lymphocyte apoptosis likely represents a part of an overall beneficial immune response (35). The role that apoptosis plays in
immune regulation and, potentially, in limitation of the pathology that would be related to the response to viral challenge warrants further investigation.
Lymphocyte depletion via apoptosis after exposure to IAV could be the
result of virus-induced cytokine stimulation, viral
induction of Fas,
or other cell-virus interactions. Further studies
will concentrate on
the abilities of specific IAV-induced cytokines
or viral proteins to
function as triggers of apoptosis. IAV is
an effective inducer of
cytokines such as gamma interferon and
TNF-
(
30), and
both factors have been shown to increase Fas
and FasL expression on T
cells (
1) and induce apoptosis (
38).
A number
of IAV gene products warrant consideration as possible
triggers of
apoptosis. Differential induction of apoptosis has
been seen in MDCK
and U-937 cells exposed to IAV strains of differing
virulence
(
27). Clone 7a (virulent for humans and ferrets) induced
more apoptosis than A/Fiji (attenuated for both species), and
the
ability of these clones to induce apoptosis was correlated
with the
differences in the amounts of neuraminidase activity
in the two strains
(
33). In this same study treatment with anti-neuraminidase
compounds was shown to abrogate apoptosis in MDCK cells
(
33).
IAV nonstructural protein nucleoprotein NS1 and NP
are also possible
candidates as triggers of apoptosis, and both are
produced early
in infection (
34). The NS1 of
A/Duck/Alberta/35/76 (H1N1) has
50% homology to regions of the Fas
antigen (
18), which could
support a role for NS1 in
induction of apoptosis. Coexpression
of Bcl-2 has been shown to alter
apoptosis, possibly through inhibition
of localization of NS1 and NP
from the nucleus to the cytoplasm
(
18). Although
production of either NA, NP, or NS1 may play
a role in induction of
apoptosis, other as-yet-undetermined viral
gene products or
intracellular processes must also be involved
and warrant
investigation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, Department of Internal Medicine, University of
Texas Medical Branch, 301 University Ave., Galveston, TX 77555-0435. Phone: (409) 747-1950. Fax: (409) 772-6527. E-mail:
jnichols{at}utmb.edu.
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Journal of Virology, July 2001, p. 5921-5929, Vol. 75, No. 13
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.73.13.5921-5929.2001
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Abstract
Introduction
