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(Journal of Leukocyte Biology. 2001;69:50-56.)
© 2001 by Society for Leukocyte Biology

Neutrophil survival is markedly reduced by incubation with influenza virus and Streptococcus pneumoniae: role of respiratory burst

Georg Engelich, Mitchell White and Kevan L. Hartshorn

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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ABSTRACT
 
Bacterial superinfections are an important cause of morbidity and mortality during influenza A virus (IAV) epidemics. We demonstrate that incubation with the combination of IAV and Streptococcus pneumoniae caused marked reductions in survival of neutrophils in vitro compared with treatment with control buffer or IAV or S. pneumoniae alone. This cooperative effect was in part mediated by acceleration of neutrophil apoptosis as evidenced by increases in annexin-V binding and caspase-3 activation. However, GM-CSF did not increase survival of neutrophils exposed to IAV and S. pneumoniae. IAV enhanced neutrophil uptake of S. pneumoniae significantly. Furthermore, the combination of IAV and S. pneumoniae caused significantly more hydrogen peroxide production than IAV or S. pneumoniae alone. This increased respiratory burst activity contributed to the diminished neutrophil survival caused by IAV and S. pneumoniae. The NADPH oxidase inhibitor, diphenyleneiodonium, significantly improved survival of neutrophils treated with IAV and S. pneumoniae. These findings may help to explain the increased susceptibility of IAV-infected patients to infections with S. pneumoniae.

Key Words: apoptosis • annexin-V • caspase • GM-CSF


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INTRODUCTION
 
Bacterial superinfections constitute a major cause of morbidity and mortality during influenza A virus (IAV) epidemics. This is particularly true among the elderly. IAV infection appears to increase susceptibility to bacterial pneumonia, otitis, and meningitis [1 ]. The most common etiological agent of bacterial pneumonia in IAV-infected subjects is Streptococcus pneumoniae [1 ]. The mechanisms through which IAV impairs host defenses against bacterial infection have not been fully elucidated. Extensive data derived from in vitro studies and animal models indicate that IAV-induced neutrophil dysfunction is an important factor predisposing bacterial infection [2 ].

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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MATERIALS AND METHODS
 
Reagents
Dextran, sodium citrate, RPMI-1640, Dulbecco’s phosphate-buffered saline (DPBS) with Ca++ and Mg++, trypan blue stain, Wright’s Giemsa stain, scopoletin, catalase, horseradish peroxidase type II, formyl-Met-Leu-Phe (fMLP), diphenyleneiodonium (DPI), propidium iodide (PI), and sodium azide were purchased from Sigma Chemical Co. (St. Louis, MO). Ficoll-Paque was obtained from Pharmacia Biotech (Piscataway, NJ). DPBS with and without added Ca++ and Mg++ was purchased from Gibco BRL (Grand Island, NY). Organic solvents were purchased from Fisher Scientific (Fairlawn, NJ).

Neutrophil isolation and culture
Neutrophils from healthy volunteer donors were isolated to >95% purity (based on Wright’s 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 hen’s 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 109–1 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 4–20% 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 Student’s paired t-test. A p value of <=0.05 was considered to indicate a statistically significant difference.


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RESULTS
 
Incubation with the combination of IAV and S. pneumoniae shortens neutrophil survival markedly in vitro
Freshly isolated neutrophils from healthy donors were incubated with control buffer or combinations of IAV and S. pneumoniae, which had been opsonized with anti-pneumococcal antiserum (see Materials and Methods). At 5, 18, and 28 h, viable neutrophil counts and percentage of viable cells were assessed based on the ability of the cells to exclude trypan blue. As shown in Figure 1 , the combination of S. pneumoniae and IAV caused a marked decline in neutrophil viability compared with neutrophils treated with control buffer or those treated with IAV or S. pneumoniae alone. The number of viable neutrophils (expressed as % of control) was already reduced significantly by the combination of IAV and S. pneumoniae after 5 h of culture (Fig. 1A) . This decline in number of viable cells preceded the development of overt cell necrosis as assessed by trypan blue dye exclusion (Fig. 1B) .



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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.

Aliquots of the neutrophils were fixed with ethanol, treated with PI, and measured for fluorescence by flow cytometry to assess the percent of cells with hypodiploid DNA (Fig. 2 ). The combination of IAV and S. pneumoniae increased markedly the percentage of neutrophils with hypodiploid DNA after 18 and 28 (but not 5) h of incubation.



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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.

IAV and the combination of IAV and S. pneumoniae increased binding of annexin-V by neutrophils
Given the marked reduction of neutrophil viability after 18 or 28 h, it was unclear to what extent this DNA fragmentation reflected apoptosis or necrosis. Measurement of annexin-V binding to neutrophils is a very sensitive means of detecting apoptosis. Therefore, we assessed whether increases in annexin-V binding could be demonstrated after 5 h of exposure of neutrophils to IAV and/or S. pneumoniae. As shown in Table 1 , IAV alone and the combination of IAV and S. pneumoniae increased the percentage of annexin-V-positive neutrophils significantly and mean flourescence of annexin-V FITC-treated neutrophils after 5 h of exposure. However, the combination of IAV and S. pneumoniae increased these values to a significantly greater extent than IAV or S. pneumoniae alone.


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  		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

To assess the extent of cell necrosis after 5 h, neutrophils were also incubated with PI, and PI-related flourescence was measured simultaneously with annexin-V FITC fluorescence. In this assay, PI was used in a different manner than in the experiments shown in Figure 2 , where neutrophils were permeabilized with ethanol to allow free diffusion of PI into the cells. In the annexin-V experiments, the neutrophils were not permeabilized. Hence, PI uptake in the annexin-V experiments is a reflection of loss of membrane integrity (hence necrosis). This assay is analogous to the trypan blue assay. As shown in Table 1 , there was some increase in the percentage of cells that took up PI in samples treated with IAV and S. pneumoniae. However, this increase was not as great as the increase in percent of annexin-V-positive neutrophils. These results indicate, therefore, that the combination of IAV and S. pneumoniae accelerates neutrophil apoptosis.

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.



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  		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

Caspase-3 is activated by IAV and S. pneumoniae
Caspase-3 activity in neutrophils was identified by using the fluorogenic substrate Ac-DEVD-AMC. AMC release was quantified by spectrofluorometry at an emission wavelength of 450 nm. DEVD-Chinese hamster ovary cells (CHO), a specific inhibitor of caspase-3-like enzymes, blocked DEVD-AMC cleavage completely (unpublished results). Increases in caspase-3 activity occurred in neutrophils treated with control buffer alone. AMC flourescence was 6.4 ± 1.2, 10.2 ± 2.6, and 15 ± 3.7 (n=8) after 5, 10, and 18 h of incubation in control media, respectively. As shown in Table 3 , treatment of neutrophils with IAV or S. pneumoniae alone or in combination induced significantly greater increases in neutrophil caspase-3 activity than control buffer alone at all of these time points. Results are expressed as percent of control activity. This effect was time-dependent with an apparent maximum increase of activity after 10 h. The combination of IAV and S. pneumoniae did not cause a significantly greater increase in caspase-3 activity than IAV alone in the time points measured in these experiments. As shown in Figure 4 , the amount of immunoreactive procaspase-3 was reduced in cytoplasm of neutrophils treated with S. pneumoniae and/or IAV compared with those treated with control buffer alone after 18 h of incubation. This result is consistent with breakdown of procaspase-3 into the active form of the enzyme.


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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.

IAV increases neutrophil uptake and H2O2 response to S. pneumoniae
S. pneumoniae was labeled with FITC, and neutrophil uptake was assessed by measuring the extent of neutrophil-associated fluorescence. As shown in Table 4 , incubation of neutrophils with IAV caused a significant increase in uptake of S. pneumoniae. In contrast, incubation of neutrophils with S. pneumoniae did not increase uptake of IAV significantly (experiments done with FITC-labeled IAV and unlabeled S. pneumoniae; unpublished results). Neutrophils treated with the combination of IAV and S. pneumoniae also produced significantly more H2O2 than cells treated with IAV or S. pneumoniae alone.


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  		Table 4. Effect of IAV on Neutrophil Uptake of S. pneumoniae or Neutrophil H2O2 Production in Response to S. pneumoniae

The respiratory burst inhibitors, DPI, enhanced survival of neutrophils treated with IAV and S. pneumoniae
In preliminary experiments, we determined that the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor DPI blocked H2O2 production fully in response to IAV and S. pneumoniae (unpublished results). As shown in Table 5 , incubation with DPI increased survival of neutrophils in vitro significantly after exposure to IAV and S. pneumoniae. DPI also caused a 64 ± 9% reduction in the percentage of hypodiploid neutrophils in cultures treated with IAV and S. pneumoniae (assessed using PI assay as in Fig. 2 ; n=5; p<0.022 comparing IAV and S. pneumoniae-treated cells with and without DPI). In contrast, addition of catalase (1 mg/ml) to cultures did not significantly reduce the amount of hypodiploid neutrophils induced by the combination of IAV and S. pneumoniae. In four experiments in which neutrophils were treated with the combination of IAV and S. pneumoniae, there were 50 ± 10% and 60 ± 14% hypodiploid neutrophils, respectively, in cultures with and without added catalase (measurements done at 28 h; p<=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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DISCUSSION
 
Pneumococcal superinfections are a major cause of morbidity and mortality during IAV epidemics. The mechanisms through which IAV infection impairs respiratory host defenses against pneumococci have not been defined fully. However, in vitro studies and animal models have shown that IAV depresses anti-microbial responses of neutrophils [2 , 14 ]. We now demonstrate that treatment of neutrophils with the combination of IAV and opsonized S. pneumoniae causes marked reduction of in vitro survival of neutrophils. The cooperative effect of these pathogens was significantly greater than effects of IAV or S. pneumoniae alone. This combined effect was demonstrated repeatedly through numerous experiments using diverse assays. We have shown similar results using the combination of IAV and E. coli [6 ]. However, the current findings are potentially of greater clinical significance because S. pneumoniae is the most common cause of bacterial superinfection in IAV-infected subjects.

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.


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ACKNOWLEDGEMENTS
 
This work was supported by NIH grant HL 58910 (to K. L. H.). We thank Alfred Tauber for critical reading of the manuscript.

Received March 30, 2000; revised September 20, 2000; accepted September 21, 2000.


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