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Am J Physiol Lung Cell Mol Physiol 289: L606-L616, 2005. First published June 10, 2005; doi:10.1152/ajplung.00130.2005
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Respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza A virus

Mitchell R. White,1 Erika Crouch,2 Jenny Vesona,1 Paul J. Tacken,3 Joseph J. Batenburg,3 Rikke Leth-Larsen,4 Uffe Holmskov,4 and Kevan L. Hartshorn1

1Department of Medicine, Section of Hematology/Oncology, Boston University School of Medicine, Boston Massachusetts; 2Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri; 3Department of Biochemistry and Cell Biology, Utrecht University Faculty of Veterinary Medicine, Utrecht, Netherlands; and 4Department of Immunology and Microbiology, University of Southern Denmark, Odense, Denmark

Submitted 23 March 2005 ; accepted in final form 5 June 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxidants and neutrophils contribute to lung injury during influenza A virus (IAV) infection. Surfactant protein (SP)-D plays a pivotal role in restricting IAV replication and inflammation in the first several days after infection. Despite its potent anti-inflammatory effects in vivo, preincubation of IAV with SP-D in vitro strongly increases neutrophil respiratory burst responses to the virus. Several factors are shown to modify this apparent proinflammatory effect of SP-D. Although multimeric forms of SP-D show dose-dependent augmentation of respiratory burst responses, trimeric, single-arm forms either show no effect or inhibit these responses. Furthermore, if neutrophils are preincubated with multimeric SP-D before IAV is added, oxidant responses to the virus are significantly reduced. The ability of SP-D to increase neutrophil uptake of IAV can be dissociated from enhancement of oxidant responses. Finally, several other innate immune proteins that bind to SP-D and/or IAV (i.e., SP-A, lung glycoprotein-340 or mucin) significantly reduce the ability of SP-D to promote neutrophil oxidant response. As a result, the net effect of bronchoalveolar lavage fluids is to increase neutrophil uptake of IAV while reducing the respiratory burst response to virus.

surfactant protein D; surfactant protein A; collectin; glycoprotein-340; bispecific protein

INFLUENZA A VIRUSES (IAVs) are a major cause of morbidity and mortality worldwide. In the United States alone influenza epidemics account for as many as 50,000 deaths per year (41). Innate immune proteins within the respiratory tract, including the pulmonary collectins, surfactant proteins D and A (SP-D and SP-A), and lung scavenger receptor-rich glycoprotein-340 (lung gp-340), have strong IAV neutralizing and aggregating activity (4, 14–16, 19, 30, 34, 35). Among the known human collectins, SP-D has the strongest in vitro IAV neutralizing and aggregating activity (19). Removal of SP-D from bronchoalveolar lavage (BAL) fluid significantly reduced antiviral activity (19). SP-D knockout mice exhibit greatly increased viral titers, lung inflammation, and illness after infection with IAV (26, 35). SP-A knockout mice also exhibit increased viral titers and inflammation after IAV infection, although these effects are less pronounced than in SP-D knockout mice (26, 34). Removal of SP-D or gp-340 from human BAL fluid or saliva reduces IAV neutralizing activity of these fluids (15, 19).

Whereas SP-D-mediated viral inhibition depends on binding of SP-D to high-mannose oligosaccharides on the viral envelope proteins, SP-A- and gp-340-mediated inhibition involves binding of the viral hemagglutinin to sialylated carbohydrates on SP-A or gp-340 (3, 15, 16). For this reason both SP-A and gp-340 have antiviral activity against strains of IAV that are resistant to SP-D (15, 16).

IAV, the collectins, and gp-340 also have interactions with phagocytic cells. The interactions of neutrophils in particular with IAV are of significance for several reasons. Early after IAV infection neutrophils infiltrate the airway probably due to release of chemokines that attract neutrophils (2, 43). Of interest, neutrophil influx is much greater after IAV infection of SP-D or SP-A knockout mice than control mice (34, 35). The role of neutrophils in clearance of IAV infection is unclear, with one study indicating that abrogation of the neutrophil response impaired recovery (10), whereas another found improved outcome (43). A recent study demonstrated that infection of mice with the IAV strain responsible for the 1918 pandemic caused marked neutrophil influx in the lung and that this effect was attributable to as yet undefined properties of the hemagglutinin (HA) molecule of that strain (31). Furthermore, highly pathogenic avian IAV strains that have recently infected humans also elicit exuberant inflammatory responses (5). These findings support the concept that excessive inflammatory responses (including exuberant neutrophil influx) may be responsible for increased lethality of some IAV strains.

IAV also causes neutrophil dysfunction and accelerated neutrophil apoptosis (6, 17, 21). The latter effect is most pronounced when neutrophils are co-incubated with IAV and bacteria. These effects appear to contribute to the predisposition of IAV-infected individuals to suffer bacterial superinfections. Bacterial superinfections constitute a major cause of morbidity and mortality during IAV epidemics (12, 24, 29). This is particularly true among the elderly. IAV infection appears to increase susceptibility to bacterial pneumonia, otitis, and meningitis.

In addition to causing neutrophil dysfunction, the virus acts as a stimulus for neutrophil respiratory burst responses through cross-linking of sialylated surface receptors on the cells mediated by the viral HA (7, 14, 21). The IAV-induced respiratory burst may contribute to killing of IAV but may also be deleterious to the host. Treating neutrophils with combinations of IAV and bacteria causes marked acceleration of neutrophil apoptosis that is mediated by the respiratory burst (6, 9). In mouse models of IAV infection, oxidant production contributes to lung injury and mortality (2).

In vitro, preincubation of IAV with collectins causes enhanced neutrophil uptake of IAV and markedly increases neutrophil respiratory burst responses to the virus (19, 22, 23). SP-D is the most potent among collectins in causing these effects (19). However, SP-D appears to play a predominantly anti-inflammatory role during IAV infection in vivo in that SP-D(–/–) mice have greatly reduced neutrophil influx and inflammatory responses compared with controls when infected with common recent human IAV strains (26, 35).

In this paper we demonstrate that SP-D can either inhibit or enhance neutrophil respiratory burst responses to IAV depending on assay conditions, that the effects of SP-D are modulated by the presence of other respiratory innate immune proteins, and that the effects of SP-D on neutrophil respiratory burst responses can be dissociated from effects on neutrophil uptake of the virus.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Materials

Dulbecco's phosphate-buffered saline with (PBS++) and without (PBS) calcium and magnesium were purchased from GIBCO-BRL (Grand Island, NY). The previously described (40) hybridoma that produces the A77 MAb directed against human CD89 was kindly provided by Dr. R. F. Graziano (Medarex, Annandale, NJ). Mucin type 1-S was obtained from Sigma (St. Louis, MO) and was derived from bovine submaxillary glands.

Virus Preparation

IAV was grown in the chorioallantoic fluid of 10-day-old chicken eggs and purified on a discontinuous sucrose gradient as previously described (17). The virus was dialyzed against PBS to remove sucrose, aliquoted, and stored at –80°C until needed. Philippines 82/H3N2 and PR-8 strains were kindly provided by Dr. E. Margot Anders (Univ. of Melbourne, Melbourne, Australia) and Dr. Jon Abramson (Wake Forest University, Winston-Salem, NC). The HA titer of each virus preparation was determined by titration of virus samples in PBS with thoroughly washed human type O, Rh(–) red blood cells as described (23). After being thawed, the viral stocks contained ~5 x 108 plaque forming units/ml.

Gp-340 and Collectin Preparations

The various innate immune proteins used in this study are summarized in Table 1. Recombinant human SP-D (rhSP-D) was produced in CHO-K1 cells as previously described (13). For these studies the dodecameric fraction of rhSP-D was used in most instances. The dodecameric fraction and high-molecular-weight multimers fraction were purified by gel filtration as described (13).


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  		Table 1. Innate immune proteins utilized in this study

 
Natural human SP-D was isolated from amniotic fluid as previously described (33). A pool of amniotic fluid (n = 6) was centrifuged at 4,000 rpm and 4°C for 30 min and purified by maltosyl agarose affinity chromatography (44). Purified SP-D was concentrated with a Vivaspin 6 (10,000 molecular weight cut off) concentrator (Vivascience, Gloucestershire, UK), and the structurally different forms of SP-D were separated by gel filtration chromatography as described (13). In brief, the samples were applied to an analytical Superose 6 column using an fast-performance liquid chromatography system (Amersham Biosciences, Freiburg, Germany) and eluted with Tris-buffered saline (TBS), pH 7.4, containing 10 mM EDTA and 0.05% emulphogen at a flow rate of 24 ml/h. Fractions of 0.8 ml were collected and quantified by an SP-D ELISA. SP-D eluted as two structurally different forms and was collected in fractions 3, 4 (high-molecular-weight SP-D) and 6, 7 (low-molecular-weight SP-D), respectively. As previously demonstrated, the high-molecular-weight fraction consists predominantly of dodecamers, whereas the low-molecular-weight fraction consists of trimers. SP-A was kindly provided by Dr. Jeffrey Whitsett (Children's Hospital and Medical Center, Cincinnati, OH).

The collectin and gp-340 preparations used in this report were tested for degree of contamination with endotoxin using a quantitative endotoxin assay (Limulus amebocyte lysate; Bio-Whittaker, Walkersville, MD). The final concentrations of endotoxin in samples containing the highest concentrations of collectins were ~20–100 pg/ml (or 6–12 endotoxin units/ml using internal assay standard). These concentrations of endotoxin showed no effect on neutrophil uptake or burst responses in our assays.

Lung gp-340 was isolated as described (28). In brief, BAL fluid was centrifuged at 300 g for 10 min and then at 10,000 g for 30 min. Subsequently, the 10,000 g and the precipitate were dissolved in TBS containing 10 mM EDTA and incubated for 18 h at 4°C with gentle stirring. Insoluble material was removed by centrifugation at 10,000 g. The supernatant was applied to a Mono-Q fast flow column (Pharmacia), and retained proteins were eluted with a linear gradient of NaCl 0–1 M. The gp-340 containing fractions were separated by gel permeation chromatography on a Superose 6 column. The purity of the resulting gp-340 was analyzed by chromatography and SDS-PAGE. The gp-340 preparation was free of SP-D and SP-A or other major contaminants as assessed by SDS-PAGE.

The bispecific recombinant homotrimeric fragment of human SP-D (rfSP-D)/anti-CD89 protein was prepared as described (45). In brief, rfSP-D, consisting of eight Gly-X-Y repeats of the collagen region, {alpha}-helical coiled-coil neck region, and carbohydrate recognition domain (CRD), produced according to Madan et al. (39), was coupled to Fab' fragment of the A77 MAb directed against CD89 using 4-(N-maleimidomethyl)cyclohexane-1-carboxylic 3-sulfo-n-hydroxysuccinimide (Pierce, Rockford, IL). Endotoxins were removed by polymyxin B treatment. Endotoxin levels of rfSP-D/anti-CD89 and the trimeric fragment rfSP-D did not exceed 18 pg/µg of protein.

Source of BAL Fluids

BAL fluids were obtained from healthy volunteer donors. Between 150 and 200 ml of normal saline were instilled for the lavage. Fluids obtained by this procedure were subjected to an initial centrifugation (150 g) to remove cells and large particulate matter. BAL fluids were obtained after informed consent as approved by the Boston University School of Medicine Institutional Review Board for Human Research.

Neutrophil Preparation

Neutrophils from healthy volunteers were isolated to >95% purity by dextran precipitation, followed by Ficoll-Paque gradient separation for the removal of mononuclear cells and then hypotonic lysis to eliminate any contaminating erythrocytes, as previously described (17). Cell viability was determined to be >98% by trypan blue staining. The isolated neutrophils were resuspended at the appropriate concentrations in control buffer (PBS++) and used within 2 h.

Measurement of IAV Uptake by Neutrophils

IAV was labeled with FITC, and viral uptake by neutrophils was assessed as previously described (16). In brief, aliquots of virus were incubated with neutrophils for 30 min at 37°C, followed by addition of 0.2 mg/ml of trypan blue to quench extracellular fluorescence, before measurement of neutrophil fluorescence by flow cytometry. In some experiments the virus was preincubated with various concentrations of BAL fluid, wild-type RhSP-D, SP-A, or gp-340 for 30 min at 37°C before addition to neutrophils. In other experiments, neutrophils were preincubated with rhSP-D dodecamers or gp-340 (30 min, followed by washing in PBS), before addition of IAV or SP-D-treated IAV.

Measurement of Neutrophil H2O2 Production

H2O2 production was measured by assessing reduction in scopoletin fluorescence as previously described (18).

Statistics

Statistical analysis was performed using the "Statmost" program (DataMost, Salt Lake City, UT). When comparing two sets of values to each other we used Student's t-test (paired, two-tailed). When comparing several experimental conditions to a single control, we performed ANOVA with Dunnett's post hoc analysis. A P value of ≤0.05 was considered statistically significant.


   RESULTS
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The Ability of SP-D to Increase Neutrophil H2O2 Responses to IAV Depends on the Sequence of Incubation of IAV or Neutrophils With SP-D and the Multimerization State of SP-D.

We have previously demonstrated that preincubation of IAV with SP-D dodecamers or higher order multimers increases IAV-stimulated neutrophil H2O2 responses (13). These findings were confirmed, as shown in Fig. 1A. However, preincubation of neutrophils with SP-D followed by addition of IAV had the opposite effect: recombinant SP-D reduced the IAV-stimulated H2O2 response (Fig. 1B). Similar results were obtained with another strain of IAV (the PR-8 strain), which is resistant to binding or inhibition by SP-D due to lack of high-mannose oligosaccharides on the viral envelope proteins (Fig. 2). These results do not appear to reflect a general effect of SP-D on neutrophil oxidant production because H2O2 responses stimulated by the bacterial peptide formyl-Met-Leu-Phe (fMLP) were not inhibited.



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  		Fig. 1. Effects of human surfactant (SP)-D on neutrophil respiratory burst responses to influenza A virus (IAV) and neutrophil uptake of IAV. A: IAV was preincubated with purified recombinant human (rh) SP-D dodecamers for 30 min followed by addition of these complexes to human neutrophils. This procedure resulted in increased H2O2 responses compared with untreated IAV. Results shown are means ± SE of 6 experiments involving different neutrophil donors and are expressed as nmol of H2O2 generated per 4 x 106 neutrophils over 3 min. The concentrations of rhSP-D (or natural SP-D in D) used are shown (in µg/ml). B: neutrophils were preincubated for 30 min with rhSP-D followed by washing and then addition of IAV. This procedure results in significant reduction of IAV-stimulated H2O2 responses (means ± SE, n = 4). C: similar preincubation of neutrophils with rhSP-D dodecamers significantly increased neutrophil uptake of FITC-labeled IAV (n = 4). Neutrophil fluorescence is given in arbitrary fluorescence units obtained by flow cytometry. D: IAV (as in A) was preincubated with high-molecular-weight (high MW, predominantly dodecameric) or trimeric forms of natural human amniotic fluid SP-D. The multimeric forms of SP-D increased H2O2 responses of neutrophils, whereas preincubation of IAV with SP-D trimers did not. *Significantly (P < 0.05) increased H2O2 response or viral uptake compared with control; **significantly reduced H2O2 responses.

 


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  		Fig. 2. Preincubation of neutrophils with rhSP-D dodecamers reduces H2O2 responses to the PR-8 strain of IAV but not to formyl-Met-Leu-Phe (fMLP). Neutrophils were preincubated with the indicated concentrations of rhSP-D dodecamers (in µg/ml, as in Fig. 1, B and C), followed by addition of either PR-8 IAV or the fMLP. Results are means ± SE of 6 experiments on separate neutrophil donors. The concentration of 2 µg/ml of SP-D significantly reduced H2O2 responses to PR-8 (P < 0.04) but not to fMLP.

 
We have previously demonstrated that preincubation of IAV with dodecameric or more highly multimerized SP-D increases neutrophil uptake of the virus (13). The increased uptake was not observed with trimeric SP-D preparations (4, 45). Of interest, preincubation of neutrophils with recombinant SP-D dodecamers also resulted in increased neutrophil uptake of the virus (Fig. 1C). Hence, preincubation of neutrophils with SP-D increased neutrophil uptake while decreasing IAV-stimulated H2O2 responses.

We have also shown previously that preincubation of IAV with trimeric recombinant SP-D preparations does not result in increased neutrophil H2O2 responses (4, 45). The experiments in Figs. 1, A–C, and 2 were performed with rhSP-D dodecamers. Natural human SP-D varies in degree of multimerization and serum concentration depending on a polymorphisms, including one involving amino acid 11 in the NH2-terminal domain of the molecule (33). Subjects homozygous for threonine at this position (17% of Danish population) have lower serum levels of SP-D and the molecule exists predominantly in a trimeric form, whereas those homozygous for methionine (35% of Danish population) have higher serum levels and the protein is predominantly present in the form of dodecamers. We isolated high-molecular-weight (predominantly dodecameric) and low-molecular-weight (predominantly trimeric) forms of SP-D from amniotic fluid and tested their ability to enhance neutrophil H2O2 generation. As shown in Fig. 1D, preincubation of IAV with the high-molecular-weight form of natural SP-D increased neutrophil H2O2 response, whereas the low-molecular-weight form did not.

Both natural and recombinant SP-D also exist in very highly multimerized forms (containing up to 32 trimeric carbohydrate recognition head domains). These highly multimerized forms can be separated from trimers or dodecamers by gel filtration (13). As shown in Fig. 3, relatively low concentrations of highly multimerized SP-D increased neutrophil H2O2 responses to IAV. This effect was dependent on the amount of multimerized SP-D when a fixed amount of IAV was used, because a higher concentration of the multimers did not in-crease the H2O2 response. A similar trend was seen with SP-D dodecamers (Fig. 1A). Of note, this higher concentration of multimers fully inhibited HA activity of the virus.



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  		Fig. 3. Relative concentration of SP-D multimers to IAV inoculum markedly affects the neutrophil oxidant response to virus. IAV was preincubated with either 0.4 or 1.6 µg/ml of rhSP-D multimers followed by assessment of neutrophil H2O2 responses. Results are means ± SE of 4 experiments (using 4 different neutrophil donors). The H2O2 response to IAV treated with 0.4 µg/ml of SP-D multimers was significantly increased (*P < 0.05), whereas response to IAV treated with 1.6 µg/ml was not increased compared with IAV alone. Mean hemagglutinin (HA) titers for the samples are shown.

 
Effects of Whole BAL Fluid and of IAV- or SP-D-binding Proteins on Neutrophil Activation by IAV

BAL fluid of healthy volunteer donors increases neutrophil uptake of IAV but does not increase IAV-stimulated H2O2 generation. Preincubation of IAV with BAL fluid increased neutrophil uptake of IAV compared with untreated virus: 40 µl of BAL fluid increased uptake of IAV to 172 ± 11% of control (n = 7, P < 0.005). Similar results were obtained with BAL fluid from a second healthy volunteer donor (e.g., increased uptake to 177 ± 24% of control, n = 3, P < 0.05).

Preincubation of IAV with BAL fluid did not increase neutrophil H2O2 responses to IAV (Fig. 4A). To determine whether these effects would be observed in the absence of large surfactant aggregates (which themselves might have depressing effects on neutrophil H2O2 responses) (1), we subjected the BAL fluid to centrifugation at 10,000 g for 30 min, which removes these aggregates and most of the SP-A in BAL fluid (25). Preincubation of IAV with the BAL supernatant also did not increase neutrophil H2O2 responses to IAV (Fig. 4A). We also tested the effect of preincubating the neutrophils with whole BAL fluid on subsequent H2O2 responses to IAV or fMLP (Fig. 4B). BAL fluid reduced H2O2 generation to IAV but did not affect the response to fMLP.



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  		Fig. 4. Normal donor bronchoalveolar lavage (BAL) fluid (BALF) reduces neutrophil H2O2 responses to IAV. A: IAV was preincubated for 30 min at 37°C with either whole BALF or the BALF supernatant obtained after centrifugation at 10,000 g. After this the viral samples were added to neutrophils, and H2O2 responses were measured. Results are means ± SE of 5 experiments with separate neutrophil donors. BALF did not significantly change the response compared with virus incubated in buffer alone (control). B: neutrophils were preincubated with whole BALF (100 µl/2 ml of PBS) for 10 min at 37°C before addition of IAV or fMLP. Results are means ± SE of 3–4 experiments and are expressed as nmol/min of H2O2 produced after stimulation with either IAV or fMLP. BALF reduced the response to IAV (as indicated) but not to fMLP.

 
Hence, BAL fluids were shown to increase neutrophil uptake of IAV but not to increase, or even to reduce, neutrophil H2O2 responses to the virus. Unlike purified SP-D, BAL fluids did not increase the IAV-induced H2O2 responses even when the virus was preincubated with BAL fluid before addition to neutrophils. This difference in the effects of purified SP-D and BAL fluids on IAV-stimulated H2O2 responses was not accounted for by large-aggregate surfactant in the BAL fluid. We next evaluated the possible effects of several other proteins in BAL fluid (i.e., SP-A, lung gp-340, and mucin) that bind to IAV and/or to SP-D to see how these modulate the effects of SP-D.

Combined incubation of IAV with SP-A and SP-D has less than additive effects on neutrophil respiratory burst responses. SP-A at a concentration of 32 µg/ml increased neutrophil uptake of IAV. No competitive effects were observed when IAV was preincubated with both SP-A and SP-D (Table 2). Preincubation of IAV with SP-A alone also caused significant increases in neutrophil H2O2 responses to IAV (Fig. 5). When the virus was preincubated with both SP-D and SP-A, however, the resulting H2O2 response was significantly reduced compared with SP-D alone.


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  		Table 2. Effect of SP-D and/or SP-A on neutrophil uptake of IAV

 


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  		Fig. 5. Combinations of SP-D and SP-A show less than additive effects on the neutrophil oxidant response. IAV was pretreated with either various concentrations of SP-D or SP-A alone or in combination as indicated. These samples were then added to neutrophils, and H2O2 generation was measured. Results are means ± SE of 3 or more experiments (involving different neutrophil donors). All concentrations of SP-D or SP-A alone or in combination significantly increased H2O2 generation compared with response to IAV alone. However, where indicated (**) combinations of SP-A and SP-D causes significantly less increase in H2O2 generation than SP-D alone.

 
Lung gp-340 does not increase neutrophil uptake of, or H2O2 responses to, IAV and counteracts the enhancing effects of SP-D on IAV uptake and IAV-stimulated H2O2 responses. We have recently reported that lung gp-340 has some cooperative antiviral effects when combined with SP-D (46). For instance, combinations of SP-D and lung gp-340 have additive effects in viral aggregation and HA inhibition assays. Gp-340 is present in alveolar macrophages, and one hypothesis is that it serves as a receptor for SP-D on phagocytic cells (28). Hence, we tested whether it would alter interactions of IAV or SP-D with neutrophils. Preincubation of IAV with gp-340 did not cause statistically significant increase in uptake of IAV (see Fig. 7). These experiments were repeated with several different preparations of gp-340 and higher doses than those shown in Fig. 6 with similar results, or with actual reduction in viral uptake (see Table 3). Preincubation of neutrophils with gp-340 also did not result in significant increase in uptake of IAV (data not shown).



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  		Fig. 7. Cooperative inhibition of HA activity of IAV by gp-340 and SP-D. HA titers measured on viral samples used in experiments shown in Fig. 6. Note that both SP-D and gp-340 alone reduced viral HA activity but that the combinations of SP-D and gp-340 caused greater inhibition. The effect of combination of SP-D and gp-340 was significantly greater than the effect of either protein alone at the same concentration. Results are means ± SE of 4 experiments.

 


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  		Fig. 6. Glycoprotein (gp)-340 decreases the ability of SP-D to increase neutrophil uptake of IAV. Values shown are means ± SE neutrophil fluorescence from 4 experiments. Neutrophils were treated with FITC-IAV alone or virus that had been preincubated with either gp-340 or rhSP-D or both (as indicated). All samples treated with SP-D (with or without gp-340) had significantly increased uptake compared with IAV treated with control buffer (P < 0.05). Gp-340 alone did not significantly increase viral uptake. However, gp-340 did significantly reduce the ability of SP-D to increase viral uptake when the virus was preincubated with both gp-340 and SP-D at 3.2 µg/ml (P < 0.04 compared with treatment with 3.2 µg/ml SP-D alone, indicated by *). Representative flow cytometry tracings from these experiments are shown on right. Tracings shown in B are neutrophils alone and neutrophils treated with unopsonized virus alone. C: virus was preincubated with either SP-D 3.2 µg/ml alone or the combination of gp-340 and SP-D (both at 3.2 µg/ml) before being added to neutrophils. Mean neutrophil fluorescence values are noted in parentheses.

 

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  		Table 3. Gp-340 does not alter the ability of SP-A to increase neutrophil uptake of IAV

 
Figure 6 demonstrates that preincubation of IAV with dodecameric SP-D markedly increases neutrophil uptake of the virus. When IAV was preincubated with combinations of gp-340 and SP-D, no significant additive effects on viral uptake were seen. In fact, the combination of 3.2 µg/ml gp-340 and SP-D resulted in significantly less neutrophil uptake of IAV than 3.2 µg/ml of SP-D alone. Note that the combination of 3.2 µg/ml gp-340 and SP-D caused complete inhibition of IAV hemagglutination activity (Fig. 7).

Preincubation of neutrophils with gp-340 followed by addition of SP-D-treated IAV also resulted in reduced uptake compared with SP-D-treated IAV alone: treatment of IAV with 3.2 µg/ml of SP-D increased uptake from 44 ± 18 to 170 ± 43 (P < 0.05, n = 3, results expressed in mean fluorescence units), whereas if neutrophils were first preincubated with 1.6 or 3.2 µg/ml of gp-340 the uptake of SP-D-treated virus was reduced by 29 ± 5 and 37 ± 16%, respectively (P < 0.01 for 1.6 µg/ml gp-340).

Gp-340 had similar effects on neutrophil H2O2 responses to IAV. IAV was preincubated with several concentrations of gp-340 alone, SP-D alone, or a combination of SP-D and gp-340. SP-D again markedly increased neutrophil H2O2 generation, whereas gp-340 did not (Fig. 8). Furthermore, the combination of gp-340 and SP-D caused markedly less H2O2 generation by neutrophils than SP-D alone. Preincubation of neutrophils with gp-340 followed by addition of IAV also resulted in less H2O2 generation than that caused by IAV alone (Fig. 9). Gp-340 did not affect H2O2 responses to fMLP.



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  		Fig. 8. Gp-340 does not increase neutrophil H2O2 generation and counteracts the increased response caused by SP-D. IAV was preincubated with lung gp-340, SP-D, or a combination of the proteins as indicated. These samples were then added to neutrophils, and H2O2 generation was measured. Results are means ± SE of 3 or more experiments. SP-D significantly increased H2O2 generation, but gp-340 did not. Addition of gp-340 in combination with SP-D caused a marked reduction in the response triggered by SP-D-treated IAV (**P < 0.05). Amounts of gp-340 or SP-D shown are in µg/ml.

 


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  		Fig. 9. Lung gp-340 and mucin reduce neutrophil H2O2 responses to IAV but not to fMLP. Results are means ± SE of 3–4 experiments and are expressed as nmol/min of H2O2 produced after stimulation with either IAV or fMLP. Neutrophils were preincubated with control buffer alone, gp-340 (1 µg/ml), or bovine submaxillary mucin (12 µg/ml) for 10 min at 37°C before addition of IAV or fMLP. No response occurred after addition of gp-340 or mucin alone. Gp-340 and mucin reduced the response to IAV (as indicated) but not to fMLP.

 
Gp-340 does not alter the ability of SP-A to increase neutrophil uptake of IAV. Because SP-A has also been reported to bind to gp-340, we tested whether gp-340 alters the ability of SP-A to increase neutrophil uptake of IAV. As shown in Table 3, gp-340 alone did not increase neutrophil uptake of IAV in this set of experiments. In fact, at the highest concentration of gp-340 used (12 µg/ml), a reduction in viral uptake occurred. SP-A (32 µg/ml) alone increased viral uptake and simultaneous incubation of IAV with this concentration of SP-A and various concentrations of gp-340 resulted in similar uptake as SP-A alone.

Mucin does not increase neutrophil uptake of IAV and reduces IAV-induced H2O2 generation. Mucins are highly sialylated glycoproteins present in oral and respiratory secretions that aggregate and inhibit HA activity of IAV (46). Preincubation of IAV with mucin did not increase viral uptake by neutrophils (99 ± 17% of control for IAV preincubated with 200 µg/ml mucin, n = 3). However, preincubation of neutrophils with mucin reduced the H2O2 responses to IAV without affecting the response to fMLP (Fig. 9). Hence, mucins are another component of respiratory secretions that reduce neutrophil oxidative responses to IAV.

Addition of rfSP-D/anti-CD89 to BAL Fluid Increases Neutrophil Uptake of and H2O2 Responses to IAV

We next wanted to determine whether the effects of BAL fluid on neutrophil responses to IAV could be modulated through engagement of additional neutrophil receptors by opsonized viral particles. The rfSP-D/anti-CD89 bispecific protein has the carbohydrate binding domain of SP-D that can attach to IAV (and other pathogens) chemically coupled to an Fab' fragment of an antibody directed against the Fc receptor for IgA present on neutrophils (CD89) (45). This bispecific protein significantly increases neutrophil uptake of IAV even in the presence of substantial concentrations of SP-D (45). rfSP-D/anti-CD89 caused significant further increase in neutrophil uptake of IAV compared with BAL fluid alone (Table 4). Addition of a trimer composed of the neck and CRD of SP-D (rfSP-DNCRD) did not increase viral uptake. Addition of rfSP-DNCRD/anti-CD89 to BAL fluid also resulted in increased H2O2 generation in response to IAV. IAV treated with BAL fluid alone produced 1.41 ± 0.5 nmol H2O2·3 min–1·4 x 106 neutrophils–1, whereas treatment of IAV with BAL fluid containing rfSP-D/anti-CD89 caused 3.0 ± 0.6 nmol H2O2 (n = 6, P < 0.008).


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  		Table 4. Addition of rfSP-D/anti-CD89 to BAL fluid increases neutrophil uptake of IAV

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It has become increasingly apparent that inflammatory responses, and specifically oxidant production, are important contributors to IAV-related illness. Neutrophils contribute prominently to the initial inflammatory response to IAV and more severe IAV infection is characterized by a more pronounced neutrophilic reaction (31, 35). One major paradox that has arisen in study of the innate defense role of surfactant collectins is that these proteins appear to play a strong anti-inflammatory role in vivo but can potentiate phagocyte activation under some circumstances in vitro. The ability of surfactant collectins to play both pro- and anti-inflammatory roles in vitro has been studied extensively in macrophages (11, 27). In this study we describe how the surfactant collectins differentially modify neutrophil respiratory burst activation in the presence of IAV. We focused our studies on SP-D and SP-D-binding proteins because in vitro and murine studies indicate that SP-D plays a pivotal role in regulating IAV replication and IAV-induced inflammation during the early phase of infection.

Our principal aims were to provide better characterization of the conditions under which SP-D increases neutrophil H2O2 responses to IAV in vitro, to determine whether the effects of SP-D on IAV-stimulated H2O2 responses are linked to its ability to increase neutrophil uptake of the virus, and to determine the effect of other SP-D or IAV-binding proteins on these responses. Our results help to clarify the apparent discrepancies between in vitro and in vivo findings regarding the effects of SP-D on inflammatory responses during IAV infection.

We demonstrate that the sequence of inhibition of SP-D with IAV or neutrophils and the degree of multimerization of SP-D strongly modify the ability of SP-D to increase IAV-stimulated neutrophil H2O2 responses. Among these results, the most striking is that preincubation of neutrophils with SP-D reduces subsequent H2O2 responses to IAV, whereas preincubating the virus with dodecameric SP-D strongly increases the H2O2 response to the virus. This result suggests that free SP-D is able to bind to inhibitory receptors on neutrophils and/or prevents the virus from binding to activating receptors. This finding is reminiscent of those of Gardai et al. (11) regarding activating and inhibiting receptors for surfactant collectins on macrophages. In those studies, activation of macrophages by LPS or apoptotic cell debris was increased if these ligands were preincubated with surfactant collectins, whereas activation was reduced if the macrophages were first incubated with the collectins followed by addition of LPS or cell debris. These divergent effects appear to result from engagement of distinct inhibitory collectin receptors [e.g., signal inhibitory regulatory protein (SIRP)-{alpha}] by the free collectins as opposed to activating collectin receptors (e.g., calreticulin/CD91) engaged by ligand-collectin complexes. Both SIRP{alpha} and calreticulin are present on neutrophils; however, their functional interactions with complexed or free surfactant collectins are unknown (8, 37, 38).

The inhibitory effect of preincubating neutrophils with SP-D on subsequent IAV-induced H2O2 responses was also observed with a strain of IAV to which SP-D binds only minimally (i.e., the PR-8 strain). This mouse-adapted IAV strain is also resistant to HA inhibitory and neutralizing effects of SP-D. This implies that binding of SP-D to IAV is not required to obtain the observed reduction in H2O2 response. However, this inhibitory effect of free SP-D was not a generalized effect on neutrophil respiratory burst responses because a different stimulus, fMLP, was unaffected.

We further evaluated the ability of SP-D to increase IAV-stimulated neutrophil H2O2 responses when the virus is preincubated with SP-D before addition to neutrophils. Consistent with previous findings (4, 45), this effect was dependent on the presence of dodecameric or multimeric forms of SP-D. In the current study, we show that natural trimeric forms of SP-D do not increase the IAV-stimulated oxidant response. This could result from the inability of trimeric forms of SP-D to cause viral aggregation. We have previously demonstrated a correlation between the ability of collectins to cause viral aggregation and their ability to increase IAV-stimulated H2O2 responses (4, 20). Similarly, large IAV particles have been shown to be more effective at stimulating respiratory burst responses of phagocytic cells than small particles (42). We have also shown that the ability of IAV alone or aggregates formed by incubation of IAV with collectins is dependent on cross-linking of sialylated neutrophil surface receptors for the viral HA (7, 14). This could account for the finding that preincubation of IAV with high relative concentrations of SP-D multimers did not enhance the response (Fig. 3), because viral HA activity was completely inhibited under these conditions. This may also account for the finding that some combinations of SP-D and gp-340 reduced IAV-induced H2O2 responses compared with the effect of IAV preincubated with SP-D alone (Fig. 8), since these two proteins in combination have cooperative effects in inhibiting viral HA activity (Fig. 7) (46).

Viral aggregation alone does not appear to account entirely for the respiratory burst enhancing effects of SP-D because gp-340 and mucin also induce viral aggregation (46) but do not increase respiratory burst responses caused by the virus. This suggests cooperative interactions between receptors for the viral HA and additional collectin receptors in triggering the enhanced H2O2 responses. Of interest, the bispecific protein rfSP-DnCRD/anti-CD89 strongly increases IAV-stimulated H2O2 responses (45) even in presence of BAL fluid (Table 4). The bispecific protein does not cause viral aggregation (data not shown). Hence, even nonaggregating forms of SP-D can potentiate respiratory burst responses to IAV if they can engage additional activating receptors (in the case of rfSP-DnCRD/anti-CD89, Fc receptors).

Studies of the effects of other respiratory proteins that interact with IAV or SP-D were carried out to better understand the effects of BAL fluid. We have previously shown that SP-D contributes strongly the HA inhibiting and neutralizing activity of BAL fluid (15, 19) with SP-A and gp-340 contributing to lesser extents. SP-D, SP-A, gp-340, and mucins all bind to IAV. In general, combining these other respiratory components with SP-D resulted in reductions in the ability of SP-D to increase neutrophil H2O2 responses to IAV. Like SP-D, SP-A appears to play an anti-inflammatory role in vivo during IAV infection, since SP-A(–/–) mice have increased neutrophil influx and proinflammatory cytokine generation during IAV infection (26, 34, 36). Like with SP-D, preincubation of IAV with SP-A increased neutrophil uptake of IAV and IAV-stimulated H2O2 responses. However, combining SP-D with SP-A caused a reduction in H2O2 responses compared with SP-D alone. This effect was to some extent dissociated from effects of the combination on viral uptake.

Lung gp-340 colocalizes with macrophages, and it was speculated that it might serve as a receptor for SP-D on phagocytes. However, we found no evidence that gp-340 alone modulated the uptake of IAV by neutrophils or acted as a bridge between neutrophils and SP-D. Furthermore, gp-340 either did not increase or reduce neutrophil H2O2 responses to IAV (the latter effect being observed when neutrophils were preincubated with gp-340). In addition, gp-340 reduced H2O2 responses to SP-D-treated IAV. This occurred despite the fact that combinations of SP-D and gp-340 have cooperative viral aggregating effects (46). Hence, gp-340 is another component of BAL fluid that could counteract respiratory burst enhancing effects of SP-D. Mucin inhibits HA activity of IAV and has cooperative viral aggregating effects when combined with SP-D (46).

It is of interest that natural trimeric SP-D did not increase neutrophil H2O2 responses to IAV because a significant percentage of subjects have the Thr/Thr11 form of SP-D, which is associated with higher levels of trimer. Of note, this genotype appears to be protective against respiratory syncytial virus (RSV)-induced bronchiolitis (32). This suggests that the trimeric form of SP-D is less likely to promote inflammatory responses during RSV infection. In a previous study we showed that the trimeric form of natural human SP-D has a reduced ability to bind to IAV compared with multimerized SP-D, but it does bind to the virus and can mediate certain antiviral effects (33). For instance, it did mediate neutralization of the virus (i.e., 720 ng/ml caused a reduction in infectious virus to 12.5 ± 2% of control, n = 4, P < 0.01). Trimeric forms of rat SP-D also mediate viral neutralization in vivo (47) and do not increase neutrophil respiratory burst responses to IAV (4). The role of SP-D polymorphisms in modulating responses to IAV infection is an important question for future study.

In conclusion, SP-D can have both activating and inhibiting effects on neutrophil respiratory burst responses to IAV depending on whether SP-D is incubated first with neutrophils or IAV and on the multimerization state of SP-D. We demonstrate that the ability of SP-D to increase neutrophil uptake of IAV can be dissociated from its ability to increase H2O2 responses under some conditions, most notably when neutrophils were preincubated with SP-D followed by addition of the virus (Fig. 1). In this setting, SP-D reduced H2O2 responses while increasing neutrophil uptake of the virus. Of interest is the observation that BAL fluid had similar effects. These results suggest that SP-D could increase viral clearance without increasing, or actually reducing, oxidant release. Other viral and/or SP-D binding components in BAL fluid tested in this study (SP-A, gp-340, and mucin) reduced the ability of SP-D to increase IAV-induced neutrophil oxidant responses. These studies provide impetus for further exploration of receptors on neutrophils for surfactant collectins and their role in lung inflammation. In addition, these studies suggest that the effects of collectins in vivo may be substantially modulated by other innate immune proteins present in mucosal secretions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-69031 (K. L. Hartshorn) and HL-29594 and HL-44015 (E. C. Crouch). J. J. Batenburg and P. J. Tacken were supported by European Commission Contract QLK2-CT-2000-00325. U. Holmskov was supported by grants from the Danish Medical Research Council (no. 9902278), the Novo Nordic Foundation, the Fifth (EC) Framework Program (contract no. QLK2000-00325), and the Benzon Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. R. F. Graziano (Medarex, Annandale, NJ) for providing the hybridoma A77.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. L. Hartshorn, Boston Univ. School of Medicine, EBRC 414, 650 Albany St., Boston, MA 02118 (e-mail: khartsho{at}bu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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