Department of Medicine and Pathology, Boston University School of Medicine, Boston, Massachusetts
Correspondence: Dr. Kevan Hartshorn, Department of Medicine and Pathology, Boston University School of Medicine, EBRC Room 414, 650 Albany Street, Boston, MA 02218. E-mail: khartsho{at}bu.edu
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Key Words: apoptosis annexin-V caspase GM-CSF
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IAV has been shown to induce apoptosis in airway epithelial cells in mice, cultured epithelial cells, and monocytes [3 4 5 ]. Recent studies from our laboratory demonstrate that IAV accelerates neutrophil apoptosis and reduces neutrophil survival in vitro [6 ]. Furthermore, when neutrophils were treated with IAV and Escherichia coli, a rapid, marked decline of neutrophil viability occurred. This decline in viable neutrophils was much greater than that observed with IAV or E. coli alone. These findings suggested that cooperative effects of bacteria and IAV on neutrophil survival could contribute to the predisposition of IAV-infected subjects for bacterial superinfection.
The current study demonstrates IAV and S. pneumoniae reduces neutrophil survival cooperatively in vitro. The effects of IAV and/or S. pneumoniae on annexin-V binding and caspase-3 activation are studied. The neutrophil respiratory burst response has been implicated as a cause of neutrophil apoptosis induced by E. coli, immune complexes, or phorbol myristate acetate (PMA) [7 8 9 ]. Therefore, we also assessed the contribution of neutrophil respiratory burst responses to diminished survival of neutrophils treated with IAV and/or S. pneumoniae.
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Neutrophil isolation and culture
Neutrophils from healthy volunteer donors were isolated to
>95% purity (based on Wrights Giemsa stain) by using dextran
precipitation, followed by a Ficoll-Hypaque gradient separation for
removal of mononuclear cells and hypotonic lysis to eliminate
contaminating erythrocytes [10
]. Cell viability after
isolation was >98%, as determined by trypan blue staining.
Virus preparation
IAV (Phillipines 82 H3N2 strain) was grown in the
chorioallantoic fluid of 10-day-old embryonated hens eggs and
purified on a discontinuous sucrose-density gradient, as previously
described [10
]. Virus stocks were dialyzed against
phosphate-buffered saline (PBS), aliquoted, and stored at -80°C
until used. Hemagglutination titers were determined by titration of
virus samples in PBS followed by addition of thoroughly washed human
type O red blood cells. After thawing, the virus stock contained about
5 x 1091 x 1010 infectious
units/ml and a protein concentration of 3.34 µg/ml by protein assay
(Bio-Rad, Hercules, CA).
Bacterial preparations
S. pneumoniae (type 23) was a gracious gift from Alan
J. Parkinson (Center for Disease Control Arctic Investigation
Laboratory, Anchorage, AK), and antiserum directed against type 23
S. pneumoniae was purchased from Dako (Carpinteria, CA).
S. pneumoniae was grown in our laboratory and fixed with
formalin for use in the experiments. S. pneumoniae
suspensions in PBS were washed three times and sonicated (for three,
20-s intervals) to eliminate bacterial aggregates prior to each
experiment. Unless otherwise indicated, S. pneumoniae was
preincubated with 32 µl/100 µl specific anti-pneumococcal
immunoglobulin G (IgG) for 30 min at 37°C and rewashed with PBS to
remove residual sodium azide.
Assessment of neutrophil viability and percentage of hypodiploid
neutrophils
Assessment of neutrophil viability was made after incubation of
the cells for 15 min with trypan blue dye. Neutrophils that did and did
not take up the dye were counted using a hemocytometer. Neutrophils
that did not take up trypan blue were counted as viable. Percent
viability of neutrophils was obtained by dividing the number of viable
cells by the total number of cells (i.e., those that did and did not
take up trypan blue). Percentage of cells with hypodiploid cells was
assessed by permeabilizing the cells and then exposing them to the
DNA-binding dye PI.
The method was performed using a slight modification of the method
descibed by Nicoletti et al. [11
]. Incubation
of neutrophils with IAV and/or bacteria was carried out as previously
described [6
]. Briefly, neutrophils (5x106
in 1 ml) were incubated with opsonized S. pneumoniae (
30
particles/neutrophil), IAV (multiplicity of infection,
20 infectious
particles/neutrophil), or a combination of IAV and the bacteria. After
1 h of incubation at 37°C, neutrophils were washed; resuspended
in RPMI 1640 supplemented with 4% autologous human serum, 20 mmol/L
HEPES buffer, 1% L-glutamine, 1% penicillin, and streptomycin, pH
7.4; and left to incubate at 37°C. At various periods of incubation
(5, 18, and 28 h), a fixed vol of cell suspension was removed from
culture and centrifuged at 200 g for 10 min, and the pellet
was fixed in 1 ml of ice-cold, 70% ethanol at 4°C for at least 60
min. Fixed cells were then washed with cold PBS with
Ca++ and Mg++ and
gently resuspended in 1 ml PI solution (40 µg/ml in PBS with
Ca++ and Mg++). The
cells were finally incubated in the dark at room temperature for 15 min
and analyzed on a FACScan 2 flow cytometer (Becton Dickinson, San Jose,
CA). Forward and side scatter of neutrophils were acquired
simultaneously. As a result of PI staining of individual cells, the red
fluorescence was collected under FL-2 and plotted against forward
scatter. The FL-2 data were registered on a logarithmic scale. Cell
debris was excluded from the analysis by appropriately raising the
forward-scatter threshold. Five thousand cells of each sample were
counted. All measurements were done under the same instrument settings
and evaluated using the Lysis II program.
Measurement of annexin-V binding
Phosphatidylserine exposure was measured by binding annexin-V
fluorescein isothiocyanate (FITC) using the protocol outlined in the
annexin-V assay kit (Pharmingen, San Diego, CA). The assay was
performed as described by Fadeel et al. [9
].
Briefly, this involved staining of the cells with annexin and PI (5
µg/ml) before analysis with a FACScan flow cytometer (Becton
Dickinson). Five thousand events were collected and analyzed using Cell
Quest software (Becton Dickinson). Low-fluorescence detritus was gated
out before analysis. PI staining in this assay was carried out to
determine the percentage of necrotic cells. This assay differed from
that described above for assessment of hypodiploid neutrophils in that
in this case, the cells were not permeabilized with ethanol prior to
addition of PI. Because neutrophils were stained as soon as possible
after isolation from culture and were not permeabilized, those cells
that took up PI had lost membrane integrity (i.e., analogous to uptake
of trypan blue).
Fluorometric analysis of caspase-3 activity
Caspase-3 activity was measured in vitro by cleavage
of the fluorogenic substrate DEVD-AMC
(Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin) by using a fluorometric
assay kit (Pharmingen). The caspase-3 aldehyde inhibitor Ac-DEVD-CHO
was used as a negative control. For preparation of cytoplasmic extracts
at 5 and 18 h of incubation, neutrophils were lysed in lysis
buffer (Pharmingen). Cell lysates and substrate were combined in a
reaction buffer (Pharmingen). AMC release was monitored in a
spectrofluorometer at an excitation wavelength of 380 nm and emission
wavelength of 440 nm. Relative AMC fluorescence was expressed as
percent of fluorescence of control cells.
Western blot
Neutrophils were washed twice in ice-cold PBS and lysed in a
buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM
ethylenediaminetetraacetate (EDTA), 0.2% bovine serum albumin (BSA),
and 1% Triton X-100 supplemented with 1 mM phenylmethylsulfonyl
fluoride (PMSF), 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM NaF,
and 10 mM ß-glycerophosphate (all from Sigma). The lysates were then
centrifuged at 14,000 g for 20 min at 4°C. The protein
content of the lysates was determined with BioRad protein assay kit
(Pierce, Rockford, IL) using BSA as a standard. Cell lysates (15 µg
total protein) were heat-denatured at 95°C for 5 min in the presence
of a loading buffer [0.0625 M Tris-base final, pH 6.8, 1.15% (w/v)
sodium dodecyl sulfate, 10% glycerol, 0.05% bromophenol blue, 0.06 M
iodoacetamide]. Samples were loaded on a precast polyacrylamide
gradient gel containing 420% acrylamide (Jule Inc., New Haven, CT)
and size-fractionated by electrophoresis. Proteins were electroblotted
onto an Immobilon-P transfer membrane (Millipore, Bedford, MA). Rabbit
polyclonal anti-human antibodies against procaspase-3 (Pharmingen)
recognizing the 32 kD precursor form of caspase-3 were added, and bound
antibodies were detected with LumiGloTM chemiluminescence system (KPL,
Gaithersburg, MD).
Assay of neutrophil H2O2 production
H2O2 production by neutrophils was
measured using the fluorescent scopoletin assay as previously described
[12
].
Measurement of bacterial or viral uptake by neutrophils
Bacterial or viral uptake was measured by incubating
FITC-labeled S. pneumoniae or IAV with neutrophils, followed
by evaluation of cell-associated fluorescence using flow cytometer as
described [13
]. Bacterial preparations were washed just
prior to use to remove unbound FITC. Fluorescence measurements were
done in the presence of trypan blue to quench extracellular
fluorescence.
Statistical methods
Statistical significance was determined using Students paired
t-test. A p value of
0.05 was considered to
indicate a statistically significant difference.
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Figure 1. Effects of IAV, S. pneumoniae, or the combination of IAV and
S. pneumoniae on neutrophil viability in vitro.
IgG-treated S. pneumoniae (100 µl in 1:100 dilution) and
IAV (10 µl) were incubated with neutrophils (5x106/ml)
as described in Materials and Methods. (A) Mean ± SE
number (n 6 experiments) of viable neutrophils counted in
cultures treated with IAV or S. pneumoniae alone or with the
combination of IAV and S. pneumoniae (as indicated) after 5,
18, or 28 h in culture. The results are expressed as percent of
control viable cells (i.e., number of viable neutrophils in IAV and/or
S. pneumoniae-treated cultures divided by viable cells in
cultures maintained in control medium alone for the same duration
x100). For cultures treated with the combination of IAV and S.
pneumoniae, viable cell counts were significantly less
(p<0.05) than for those treated with control medium, IAV,
or S. pneumoniae alone at all time points. (B) Percentage of
cells excluded trypan blue in cultures treated with control medium
alone, IAV, and/or S. pneumoniae. The results were obtained
by dividing the number of cells that excluded trypan blue by the total
number of cells in each culture. At 18 or 28 h of incubation, the
percentage of neutrophils excluding trypan blue was reduced
significantly in cultures treated with the combination of IAV and
S. pneumoniae compared with those treated with control
medium or IAV or S. pneumoniae alone.
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Figure 2. Effects of IAV and/or S. pneumoniae on the percentage of
hypodiploid neutrophils in culture. Results are expressed as percentage
of cells with hypodyploid DNA as measured by the PI assay and represent
the mean ± SE of five separate experiments. Asterisks
indicate where the combination of S. pneumoniae and IAV
caused significantly (P<.05) increases in hypodiploid
neutrophils compared with control buffer, IAV, or S.
pneumoniae alone.
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View this table: [in a new window] |
Table 1. Binding of Annexin-V FITC and Uptake of PI by Neutrophils Treated with
Control Medium, IAV, S. pneumoniae, or the Combination of
IAV and S. pneumoniae for 5 h
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Granulocyte-macrophage colony-stimulating factor (GM-CSF) did not
enhance survival of neutrophils treated with the combination of IAV and
S. pneumoniae
GM-CSF has been shown to delay spontaneous neutrophil apoptosis
and prolong neutrophil survival in vitro. We tested whether
GM-CSF altered the percent of hypodiploid neutrophils (Fig. 3
) or enhanced neutrophil survival (Table 2
) in samples treated with IAV and/or S. pneumoniae. As
expected, GM-CSF increased the survival of control neutrophils
significantly (i.e., neutrophils maintained in control medium alone
without IAV or bacteria; see Table 2
). GM-CSF also reduced the
percentage of hypodiploid cells in control neutrophil cultures (Fig. 3)
. However, GM-CSF did not improve survival significantly or reduce
the percentage of hypodiploid cells in neutrophil samples incubated
with the combination of IAV and S. pneumoniae.
![]() View larger version (24K): [in a new window] |
Figure 3. GM-CSF did not reduce the percentage of hypodiploid neutrophils in
samples treated with IAV and S. pneumoniae. Neutrophils
(5x106/ml) were incubated for 1 h at 37°C with
GM-CSF (100 ng/ml) alone or in combination with IAV and/or S.
pneumoniae. Neutrophils treated with GM-CSF alone (i.e., no IAV or
bacteria) had significantly less hypodiploid neutrophils than those
treated with control buffer alone (#). However, GM-CSF did not
significantly reduce the percentage of hypodiploid cells in cultures
treated with IAV and S. pneumoniae.
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Table 2. Effect of GM-CSF on Survival of Neutrophils Treated with IAV or the
Combination of IAV and S. pneumoniae
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Table 3. Caspase-3 Activity in Neutrophils Treated with IAV, S.
pneumoniae, or the Combination of IAV and S. pneumoniae
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Figure 4. IAV and S. pneumoniae reduced immunoreactive procaspase-3 in
neutrophil cytoplasm. Western blot analysis was performed on protein
extracted from cytoplasm using antibody directed against procaspase-3.
Protein bands were detected at 32 kD, matching procaspase-3 size. A, B,
C, and D indicate procaspase-3 bands from neutrophils treated,
respectively, with control buffer, IAV, S. pneumoniae, or
the combination of IAV and S. pneumoniae. After 18 h of
incubation, S. pneumoniae-treated and S.
pneumoniae and/or IAV-treated cells have less expression of the
precursor form of caspase-3 compared with control. Results are
representative of three experiments.
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Table 4. Effect of IAV on Neutrophil Uptake of S. pneumoniae or
Neutrophil H2O2 Production in Response to
S. pneumoniae
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0.08). |
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Table 5. DPI Increased Survival of Neutrophils Treated with the Combination of
IAV and S. pneumoniae
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Our findings suggest that the rapid decline in viable neutrophils in samples treated with the combination of IAV and S. pneumoniae is at least in part caused by acceleration of neutrophil apoptosis. After 28 h, a high percentage of neutrophils treated with the combination of IAV and S. pneumoniae was necrotic based on uptake of trypan blue. Hence, at this time point, it was difficult to conclude whether the presence of hypodiploid cells detected using the PI assay represented apoptosis or necrosis. However, it is interesting that the decline in viable cell numbers at earlier time points (see Fig. 1A ; especially at 5 h) preceded the development of necrosis. The annexin-V binding assay has the advantage that it can be performed on nonpermeabilized cells and hence allows a clearer differentiation of apoptotic cells [15 ]. Annexin-V binding was elevated significantly in neutrophils exposed to the combination of IAV and S. pneumoniae after 5 h in culture (Table 1) . Also, the percentage of annexin-V binding neutrophils exceeded the percentage of necrotic neutrophils at this time (Table 1) . These results suggest that apoptosis preceded necrosis in these cultures.
GM-CSF is present in lung secretions during IAV infection [16 ] and plays an important role in regulating phagocyte activation in this setting [17 ]. GM-CSF delays spontaneous neutrophil apoptosis significantly [18 ]. GM-CSF has not been found to reduce rate or severity of pneumococcal superinfections in IAV-infected animals [19 ]. In our experiments, GM-CSF enhanced survival of uninfected neutrophils (Table 2 ; Fig. 3 ). However, GM-CSF did not significantly improve survival of neutrophils treated with the combination of S. pneumoniae and IAV.
We attempted to clarify the mechanism through which IAV and/or bacteria accelerate neutrophil apoptosis. We have shown previously that IAV enhances neutrophil Fas and Fas ligand expression. Caspases, a family of cytoplasmic proteases, are involved in constitutive or Fas-mediated apoptosis [9 , 20 ]. We now demonstrate that IAV and S. pneumoniae accelerate activation of caspase-3 in neutrophils compared with the activation observed in neutrophils maintained in control medium alone. However, caspase activation alone may not account for the cooperative effects of IAV and S. pneumoniae on neutrophil survival, because there did not appear to be additive enhancement of caspase-3 activation when IAV and S. pneumoniae were added together.
Fadeel et al. [9 ] have found that PMA induces neutrophil apoptosis without stimulating caspase-3 activation. However, when NADPH oxidase activation was inhibited by addition of DPI, caspase-3 activation did occur. These findings raised the possibility that there are caspase-independent pathways of neutrophil apoptosis that involve respiratory burst activation [9 ]. We demonstrate that IAV significantly increased neutrophil uptake of, and respiratory burst responses to, S. pneumoniae. We have shown previously similar results when coincubating neutrophils with IAV and E. coli [6 ]. Cooperative binding of IAV and S. pneumoniae in Hep-2 cells has been associated with IAV-induced expression of the cell-surface antigens CD14 and CD18 [21 ]. We have shown previously that treatment of neutrophils with IAV enhances neutrophil adhesion to serum-coated surfaces and causes marked upregulation of CD11b, CD11c, and the carcinoembryonic antigen (CEA)-related antigens, CD66 and CD67 [22 , 23 ], which could account for the ability of IAV to increase neutrophil uptake of bacteria.
We considered it possible, therefore, that the ability of IAV to potentiate neutrophil uptake of, or respiratory burst responses to, S. pneumoniae and E. coli accounts in part for the cooperative effects of IAV and these bacteria on neutrophil survival. Previous studies have shown that neutrophil apoptosis induced by phagocytic substrates and other activating stimuli may be mediated by neutrophil respiratory burst responses [8 , 9 , 24 25 26 ]. Fadeel et al. [9 ] have demonstrated that DPI blocks phosphatidylserine (PS) exposure in PMA-treated neutrophils. Antioxidants have also been shown to improve the survival rate of influenza virus-infected mice [27 ]. In our experiments, DPI inhibited H2O2 production and increased survival of neutrophils treated with the combination of IAV and S. pneumoniae. DPI also significantly reduced the percentage of hypodiploid cells in these cultures. We have also found that DPI enhances survival of neutrophils exposed to IAV and E. coli (unpublished results). In contrast, catalase did not significantly reduce the percentage of hypodiploid cells in cultures treated with IAV and bacteria. This discrepancy may be explained by the fact that DPI is cell-permeant, and catalase is not. We have demonstrated previously that the respiratory burst response elicited by IAV occurs predominantly at an intracellular location [28 ]. Hence, the ability of DPI to permeate the cell membrane may have made it more effective at enhancing survival of neutrophils treated with IAV and S. pneumoniae.
In conclusion, the combination of IAV and S. pneumoniae caused markedly greater reduction in neutrophil survival in vitro than control media or IAV or S. pneumoniae alone. This effect was at least in part mediated by acceleration of neutrophil apoptosis as evidenced by increased annexin-V binding and caspase-3 activation. Respiratory burst activation also appeared to contribute importantly to decreased survival of neutrophils exposed to IAV and S. pneumoniae, as evidenced by the protective effects of DPI. Whyte et al. [29 ] have demonstrated that apoptosis is associated with decline in functional responsiveness of neutrophils. Hence, acceleration of apoptosis could account for diminished numbers and function of neutrophils in vivo in subjects infected with IAV and bacteria. These findings may in part account for the susceptibility of IAV-infected subjects for bacterial superinfection. Further studies using in vivo models would be needed to verify this hypothesis.
Received March 30, 2000; revised September 20, 2000; accepted September 21, 2000.
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