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Surfactant Protein-D Enhances Phagocytosis and Pulmonary Clearance of Respiratory Syncytial Virus

Divisions of Pulmonary Biology, Critical Care Medicine, and Pulmonary Medicine, Allergy, and Clinical Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; and Department of Pathology, Washington University School of Medicine, St. Louis, Missouri

Address correspondence to: Ann Marie LeVine, M.D., Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology and Critical Care Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail: annmarie.levine{at}cchmc.org

	Abstract

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Surfactant protein (SP)-D gene targeted (SP-D^–/–) and wild-type mice were infected with respiratory syncytial virus (RSV) by intratracheal instillation. Decreased clearance of RSV was observed in SP-D^–/– mice. Deficiency of SP-D was associated with increased inflammation and inflammatory cell recruitment in the lung after infection. In vitro, SP-D bound RSV-infected Vero cells. Binding was inhibited with ethylenediamine tetraacetic acid and maltose, suggesting that the carbohydrate recognition domain of SP-D recognizes RSV glycoproteins in a calcium-dependent manner. SP-D bound specifically to the RSV proteins G and F. Phagocytosis of RSV by alveolar macrophages was reduced in the absence of SP-D in vivo, and SP-D enhanced phagocytosis of RSV by alveolar macrophages and neutrophils but not peritoneal macrophages in vitro. Oxygen radical production by alveolar macrophages from SP-D^+/+ and SP-D^–/– mice was decreased after RSV infection, and SP-D ameliorated the inhibitory effects of RSV on oxygen radical production by macrophages and neutrophils in vitro. Because the airway is the usual portal of entry for RSV and other respiratory pathogens, the local production of SP-D is likely to play a role in innate defense responses to inhaled viruses.

Abbreviations: bronchoalveolar lavage, BAL • BAL fluid, BALF • carbohydrate recognition domain, CRD • 2,7 dichlorofluorescin diacetate, DCF • ethylenediamine tetraacetic acid, EDTA • enzyme-linked immunosorbent assay, ELISA • Eagle's minimal essential media, EMEM • fluorescein isothiocyanate, FITC • influenza A virus, IAV • interferon-, IFN- • macrophage inflammatory protein, MIP • phosphate-buffered saline, PBS • respiratory syncytial virus, RSV • surfactant protein, SP • tumor necrosis factor-, TNF-

	Introduction

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Surfactant protein (SP)-D is a member of the collectin subgroup of the mammalian C-type lectins that includes SP-A, mannose-binding protein, and conglutinin (1, 2). The collectins mediate innate host defense against various bacterial and viral pathogens. The collectins form multimeric structures resembling C1q (the first component of the complement cascade), consisting of multimeric collagenous aminoterminal domains and globular carboxyterminal, carbohydrate-binding domains (2). The C-type lectins bind carbohydrate surfaces of many microorganisms mediating phagocytosis and killing by phagocytic cells (3).

In the lung, SP-D is produced primarily by alveolar Type II cells and nonciliated bronchiolar cells (4). SP-D binds to alveolar macrophages (5), binds many species of Gram-negative bacteria, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Haemophilus influenzae (6), and binds certain Gram-positive organisms, including strains of Streptococcus pneumonia and Staphylococcus aureus (7). SP-D inhibits the infectivity of influenza A virus (IAV), induces aggregation of IAV particles, and enhances binding and uptake of IAV by neutrophils (8, 9). In vivo, SP-D–deficient mice (SP-D^–/–) show decreased viral clearance and enhanced inflammation in the lung after challenge with IAV (10). SP-D also interacts with other viruses; bovine SP-D inhibits the infectivity of rotaviruses through calcium-dependent attachment to the major viral envelope glycoprotein (11).

Respiratory syncytial virus (RSV) produces an upper respiratory tract disease that may progress to acute bronchiolitis or interstitial pneumonia. Approximately 1 in 100 children infected with RSV will require hospitalization (12); as many as 11% of hospitalized infants may require intensive care (13). Children with respiratory failure from RSV bronchiolitis have decreased concentrations of SP-A and SP-D in bronchoalveolar lavage (BAL) fluid (BALF) (14). Conditions predisposing to severe RSV disease include prematurity, bronchopulmonary dysplasia, and cystic fibrosis, conditions that may also be associated with decreased SP-D in the lung (15, 16). RSV is an enveloped, negative-strand RNA virus with a genome that encodes at least 10 viral proteins with 2 major surface glycoproteins (G and F), which are incorporated in the virus envelope. The G protein is responsible for attachment of the virus to the host cell surface (17), whereas the F protein is responsible for penetration of the virus into the host cells and subsequent cell-to-cell spread (18).

Specific as well as nonspecific immune mechanisms take part in RSV immunity and probably also RSV pathogenesis. The specific immune response includes cytotoxic T cells and antibody production. Nonspecific immune responses include natural killer cells, macrophages, neutrophils, eosinophils, and basophils (19). Cytotoxic T lymphocytes are thought to mediate clearance of virus by direct cytolysis of virus-infected cells (20). Defects in neutrophil and monocyte, chemotactic, oxidative, and bacterial killing functions have been documented in IAV infection (21, 22). In vitro, neutrophil dysfunction resulting from IAV exposure is diminished when the virus is preincubated with SP-D (8).

In spite of considerable in vitro evidence that SP-D is involved in host defense, its role in vivo has only recently been demonstrated. SP-D^–/– mice have increased inflammatory responses in the lung with bacterial infection and after IAV infection (10, 23). Although clearance of most bacteria from the lung was maintained, SP-D^–/– mice were highly susceptible to IAV infection, clearing the virus poorly. In the present study, SP-D^–/– mice were infected intratracheally with RSV. Viral clearance, lung inflammation, phagocytosis, and oxygen radical production by phagocytic cells were compared in SP-D^–/– mice and SP-D^+/+ mice, in vivo.

	Materials and Methods

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Animal Husbandry
SP-D^–/– mice were generated by targeted gene inactivation as previously described (23). NIH Swiss Black^+/+ and SP-D^–/– mice were studied. Animals were housed using barrier containment and studied under Institutional Animal Care and Use Committee-approved protocols in the animal facility of the Children's Hospital Research Foundation, Cincinnati. Routine viral serology of sentinel mice was used to assure viral free containment. Male and female mice of 20–25 g (56–63 d old) were used.

Preparation of RSV
HEp-2 cells were maintained in Eagle's minimal essential media (EMEM) supplemented with glutamine, amphotericin, streptomycin, penicillin G, and 10% low immunoglobulin fetal bovine serum (10% EMEM). The A2 strain of RSV was plaque-purified three times under agarose. The third plaque was inoculated into a subconfluent HEp-2 cell monolayer. After adsorption for 1 h at room temperature 10% EMEM was added and the infection was allowed to proceed for 3 d at 37°C until the entire monolayer showed cytopathic effects. The contents of the flask were resuspended and distributed in 1-ml aliquots, quick-frozen with alcohol/dry ice, and stored at –80°. Virus was derived from this master stock by infecting subconfluent HEp-2 monolayers at multiplicity of infection of 0.1, and harvesting the monolayer when it appeared to be completely infected. The cells and media (50 ml) were sonicated (Ultrasonic homogenizer; Cole-Parmer Instrument Co., Chicago, IL) on ice with eight 1-s bursts using output of 50. The suspension was clarified by centrifugation at 1,800 RPM for 10 min. The supernatant was frozen and stored at –80°C and thawed rapidly at 37°C for use.

Viral Infection
Administration of RSV into the respiratory tract of the mice was performed by intratracheal inoculation of 10⁷ plaque-forming units (pfu) diluted in 10% EMEM. Intratracheal inoculation as previously described (23) was used to deliver RSV. Controls received sterile 10% diluent. After inoculation, mice recovered and were returned to water and food ad libitum.

Clearance of RSV
Quantitative RSV cultures were performed 3 and 5 d on lung homogenates after inoculation of the mice with RSV. The entire lung was removed, homogenized in 2 ml of sterile 10% EMEM, weighed, and placed on ice. HEp-2 monolayers, 80% confluent in Costar 12-well plates, were used for plaque assay. Lung tissues were clarified, serially diluted in 10% EMEM, plated on HEp-2 monolayer (50 µl), adsorbed for 1 h, and 0.75% methylcellulose in 10% EMEM added to each well. After 5 d at 37°C the monolayers were fixed with 10% formalin phosphate, stained with hematoxylin-eosin, and viral plaques counted under a dissecting microscope. The resulting titer was divided by the lung weight and reported as pfu/gram of lung.

BAL
Lung cells were recovered by BAL. Animals were killed as described for viral clearance and lungs were lavaged three times with 1 ml of sterile phosphate-buffered saline (PBS). The fluid was centrifuged at 2,000 RPM for 10 min, resuspended in 600 µl of PBS, and total cells stained with trypan blue and counted under light microscopy. Differential cell counts were performed on cytospin preparations stained with Diff-Quick (Scientific Products, McGaw Park, IN).

Pathology
Lungs were inflated via a tracheal cannula at 20 cm of pressure with 4% paraformaldehyde and removed en-block from the thorax. Lungs were dehydrated and embedded in paraffin. Each lobe of the lung was bisected and four sections obtained in a caudal to cranial orientation from the cut surface. Tissue sections (5 µm) were stained with hematoxylin-eosin. Tissue sections were scored for inflammation by two observers blinded to treatment. The scoring system: 0 = no inflammation, 1 = few monocytes/macrophages, 2 = increased cells, 3 = most alveoli or bronchioles with inflammatory cells, 4 = consolidation of alveoli or intraluminal inflammatory cells.

Cytokine Production
Lung homogenates were centrifuged at 2,000 RPM and the supernatants stored at –20°C. Tumor necrosis factor- (TNF-), interleukin (IL)-1ß, IL-6, macrophage inflammatory protein (MIP)-2, and interferon (IFN)- were quantitated using murine sandwich enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer's directions. All plates were read on a microplate reader (Molecular Devices, Menlo Park, CA) and analyzed with the use of a computer-assisted analysis program (Softmax; Molecular Devices). Only assays having standard curves with a calculated regression line value > 0.95 were accepted for analysis.

Recombinant Rat SP-D
Recombinant rat SP-D dodecamers were expressed by stably transfected CHO-K1 cells as previously described (24). Secreted SP-D was isolated by maltosyl-agarose affinity chromatography and gel filtration chromatography. Bound proteins were eluted in HEPES-buffered saline containing 10 mM ethylenediamine tetraacetic acid (EDTA) and stored at –80°C. The protein concentration was estimated using a dye binding assay with bovine serum albumin as standard. The level of endotoxin contamination was quantified using an end-point chromogenic microplate assay (Chromogenix, Milan, Italy) with Escherichia coli 0111:B4 endotoxin as a standard. The endotoxin content of the purified recombinant proteins used for these experiments was < 2 ng/ml for stock solutions. Biotinylation of recombinant rat SP-D was labeled at primary amino-groups with EZ-link-Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL). Reactions were performed with limited modifications of the manufacturer's recommendation procedure as described below. SP-D was diluted in 10–20 µg/ml with PBS, pH 7.4. Sulfo-NHS-Biotin (1 mg/ml) was prepared fresh in distilled water and 10–20 µl added to the protein. Reactants were mixed, transferred to a 500-µl Slide-A-Lyser cassette (Pierce), incubated for 2 h at room temperature, and then the cassette was dialyzed against PBS for 2 h. Biotinylation was determined by the HABA/Avidin reaction and the biotinylated protein stored at –80°C. The specific activity was 50 pmole/µg SP-D.

Binding of SP-D to RSV
Vero cells (ATCC, Manassas, VA) were cultured in MEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine at 37°C with 5% CO₂. Cells were infected with RSV at a multiplicity of infectivity (MOI) of 5, incubated for 24 h at 37°C, scraped, and washed twice with phenol red–free MEM with 2 mM CaCl₂. RSV-infected cells were then incubated with 100 ng/ml of biotinylated SP-D, SP-D (100 ng/ml) with 10 mM EDTA, or SP-D (100 ng/ml) with 167 mM maltose for 1 h at 37°C. Cells were washed with MEM, incubated with streptavidin R-phycoerythrin for 30 min, washed, and binding assessed using a FACScalibur flow cytometer (BD Bioscience, San Jose, CA). Surface expression of RSV G or F proteins were determined by incubating the RSV-infected vero cells with an anti-RSV protein-G antibody (1:500 dilution) or anti-RSV protein-F antibody (1:500 dilution; Research Diagnostics, Flanders, NJ) for 1 h and staining with 1 µg/ml of fluorescein isothiocyanate (FITC)-goat anti-mouse IgA antibody (PharMingen, San Diego, CA). Cells were analyzed by flow cytometry and RSV infectivity determined by the percent increase in mean fluorescence intensity over isotype control. Recombinant vaccinia viruses expressing the attachment (RSV-G) glycoprotein of RSV (VG) and fusion (RSV-F) glycoprotein (VF) were generated as previously described (25). Vaccinia virus expressing a ß galactosidase gene (VSC11) was used as control. Vero cells were infected with vaccinia virus at an MOI of 10 for 24 h, then binding of SP-D was determined as described for RSV-infected cells. Baseline binding of SP-D to control vaccinia without VF or VG was subtracted from binding to vero cells infected with VF or VG and the results reported as fold increase in mean fluorescence intensity over control virus. Binding of SP-D to RSV-infected cells was also determined after competitive inhibition with F and G antibodies.

FITC Labeling of RSV
FITC stock was prepared at 1 mg/ml in 1 mol/L sodium carbonate, pH 8.0. FITC-labeled virus (RSV) was prepared by incubating concentrated virus stocks with FITC (10:1 mixture by volume of virus in PBS with FITC stock) for 1 h, followed by dialysis of the mixture for 18 h against PBS.

Phagocytosis of RSV
To determine if SP-D enhanced phagocytosis of RSV in vitro, alveolar macrophages, peritoneal macrophages, and neutrophils were compared for the ability to phagocytose RSV in the presence and absence of SP-D. Alveolar macrophages were obtained from wild-type mice as describe for BAL cells. Peritoneal macrophages were obtained from wild-type mice by lavaging the peritoneum five times with 5 ml of MEM media. Neutrophils were obtained after injection of the peritoneal cavity with 1 ml of 3% thioglycolate 24 h and again 4 h before peritoneal lavage. Cells were incubated for 2 h with FITC-RSV or FITC-RSV pre-opsonized with SP-D (100 ng/ml) at an MOI of 5. Cells were then washed, extracellular fluorescence quenched with trypan blue (0.02 µg/ml), and cells were washed again and resuspended in PBS with 1% paraformaldehyde. Intracellular fluorescence was determined by flow cytometry and reported as percent phagocytosis.

Phagocytosis of RSV by alveolar macrophages in vivo was measured by intratracheally infecting mice with FITC-RSV followed by evaluation of cell-associated fluorescence by flow cytometry. Two hours after infection, BAL macrophages were obtained as described for BAL, extracellular fluorescence quenched with trypan blue, and cells washed three times and resuspended in PBS containing 1% paraformaldehyde. Cell-associated fluorescence was measured on a FACScalibur flow cytometer, 10,000 cells were counted and the results expressed as percent phagocytosis.

Oxygen Radical Production
Oxygen radical production by peritoneal macrophages and neutrophils was assessed after RSV infection in the presence and absence of SP-D, in vitro. Peritoneal macrophages and neutrophils were obtained as described for the phagocytosis assay, incubated with RSV at an MOI of 5 for 24 h, then loaded with 1 mM lucigenen (Sigma, St. Louis, MO) for 5 min. Oxygen radical production from 1 x 10⁵ cells was assessed for RSV infected cells, RSV+PMA (phorbol myristate acetate) (100 ng/ml), RSV + SP-D (10–400 ng/ml), and RSV + SP-D + PMA. SP-D and PMA were added simultaneously and luminescence measured for 30 min on a Berthold model Autolumat LB 953 luminometer (Berthold Technologies, Oak Ridge, IN) and results reported as the total luminescence.

Oxygen radical production by alveolar macrophages was determined 5 d after intratracheal RSV infection of SP-D^+/+ and SP-D^–/– mice. Alveolar macrophages were obtained by BAL as described, loaded with 1 µm 2,7 dichlorofluorescin diacetate (DCF; Molecular Probes, Eugene, OR) at 37°C for 30 min, washed twice with PBS, and intracellular fluorescence measured by flow cytometry and reported as the mean fluorescent intensity for 10,000 events.

Western Blot
Western blot analysis for SP-A was performed on tissue homogenates. Lung tissue was homogenized in PBS (500 µl), spun at 500 RPM then the pellet resuspended in 1 ml 10 mM Tris-Cl (pH 7.4), 0.25 M sucrose, 2 mM EDTA, 1 mM PMSF, 10 µM leupeptin, and 10 µM pepstatin A. The sample was centrifuged at 15,000 RPM for 15 min at 2°C and pellet resuspended in the above buffer (without sucrose) and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10–27% gradient gels. Proteins were transblotted to PVDF membranes (Bio-Rad, Inc., Hercules, CA) and blocked with 10 mM Tris-Cl (pH 7.4), 0.15 M NaCl, 0.1% Tween-20 (TBS-T) containing 5% bovine serum albumin. SP-A was detected with guinea pig anti-rat SP-A serum (18) and horseradish peroxidase–conjugated secondary antibody (Calbiochem, Inc., San Diego, CA). Membranes were rinsed and developed using enhanced chemiluminescence detection reagents (Amersham, Arlington Heights, IL). Immunoreactive bands were identified by exposing the membranes to XAR film (Kodak, Rochester, NY).

Surfactant Protein D Concentrations
Concentrations of SP-D in lung homogenates were determined with an ELISA. Three and 5 d after infection with RSV, lungs from infected and uninfected wild-type mice were removed and homogenized in 2 ml of PBS. Surfactant protein D concentrations were measured in a double antibody ELISA using rabbit (AB3434; Chemacu, Temecula, CA) and guinea pig anti–SP-D antisera. Each assay plate included a standard curve generated with purified mouse SP-D. All samples were run in duplicate and the concentrations of the samples were calculated by graphing absorbance versus concentrations of controls.

Statistical Methods
Lung viral titers, BAL total cells, cytokines, phagocytosis, and oxygen radical production were compared using ANOVA and Student's t test. Lung histology was compared using Mann-Whitney Rank Sum Test. Findings were considered statistically significant at probability levels < 0.05.

	Results

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Pulmonary Pathology after RSV Administration
Intratracheal administration of RSV (10⁷ pfu) was well tolerated and all animals survived the study period. No alterations in activity or physical appearance of the animals were detected throughout the study with RSV infection. SP-D^–/– mice had increased total cells counts in BAL fluid 3 and 5 d after RSV infection (Figure 1). Baseline total cell counts in BAL fluid from controls inoculated with EMEM were 9.6 x 10⁴ ± 1.1 x 10⁴ and 1.7 x 10⁵ ± 1.4 x 10⁴ for the SP-D^+/+ and SP-D^–/– mice, respectively, mean ± SEM. Significantly greater percentages of polymorphonuclear leukocytes were detected in BALF from SP-D^–/– compared with SP-D^+/+ mice 3 and 5 d after infection (Figure 1). Lung tissue was examined by two observers blinded to treatment and scored for inflammation. Alveolar and bronchiolar inflammation was greater in the lungs of SP-D^–/– compared with SP-D^+/+ mice 3 and 5 d after RSV infection (Table 1). SP-D^–/– mice inoculated with sterile media had baseline inflammation and emphysema as previously described (26).

View larger version (20K): [in this window] [in a new window]   Figure 1. Increased lung inflammation in SP-D–/– mice after RSV infection. Lung cells were recovered by BAL, stained with trypan blue, and counted under light microscopy. Cytospin preparations of BALF were stained with Diff-Quik to identify macrophages, lymphocytes, and polymorphonuclear leukocytes. (A) SP-D–/– (open bars) mice had increased total cell counts in BAL fluid 3 and 5 d after RSV infection compared with SP-D+/+ mice (hatched bars). (B) The percentage of neutrophils in BALF was significantly greater 3 and 5 d after administration of RSV to SP-D–/– compared with SP-D+/+ mice. Data are mean ± SEM with n = 10 mice per group, *P < 0.05 compared with SP-D+/+ mice on the same day.

View larger version (11K): [in this window] [in a new window]   Figure 2. Increased viral titers in lung homogenates from SP-D–/– mice. RSV titers were determined by quantitative plaque assays of lung homogenates. Viral titers of RSV were significantly greater 3 and 5 d after administration of 107 pfu RSV in SP-D–/– (open bars) compared with SP-D+/+ (hatched bars) mice. Data are mean ± SEM with n = 15 mice per group. *P < 0.05 compared with SP-D+/+ mice.

View larger version (18K): [in this window] [in a new window]   Figure 3. Proinflammatory cytokines are increased in SP-D–/– mice after RSV infection. Concentrations of proinflammatory cytokines were assessed in lung homogenates. Increased concentrations of TNF-, IL-6, IL-1ß, and MIP-2 were found in lung homogenates from SP-D–/– (open bars) compared with SP-D+/+ (hatched bars) mice 3 and 5 d after RSV infection. Data are expressed as pg/ml and represent mean ± SEM with n = 10 mice per group. *P < 0.05 compared with SP-D+/+ mice on the same day.

View larger version (34K): [in this window] [in a new window]   Figure 4. SP-D enhanced phagocytosis of RSV. Phagocytosis of FITC-RSV was measured by flow cytometry. (A) In vitro, SP-D enhanced uptake of RSV by alveolar macrophages (A MAC) and polymorphonuclear leukocytes (PMN) but not peritoneal macrophages (P MAC). Solid bars, RSV; cross-hatched bars, RSV + SP-D. (B) Phagocytosis of RSV was greater for alveolar macrophages from SP-D+/+ (hatched bars) compared with SP-D–/– (open bars) mice 2 h after infection. Data are expressed as % phagocytosis and represent mean ± SEM. A, n = 3 mice per group. *P < 0.05 compared with phagocytosis of RSV alone. B, n = 8 mice per group. *P < 0.05 compared with SP-D+/+ mice.

View larger version (49K): [in this window] [in a new window]   Figure 5. SP-D enhanced oxygen radical production from phagocytic cells after RSV infection. Extracellular oxygen radical production by peritoneal macrophages and neutrophils was measured with lucigenin after RSV infection in vitro. Oxygen radical production was similar for peritoneal macrophages (A) and neutrophils (B) infected with RSV in the absence (white bars) or presence (black bars) of SP-D. RSV infected macrophages (hatched bars) and neutrophils (hatched bars) stimulated with PMA generated less oxygen radicals compared with uninfected cells (cross-hatched bars). However, in the presence of SP-D, RSV infected macrophages (stippled bars) and neutrophils (stippled bars) stimulated with PMA generate similar amounts of oxygen radicals as stimulated uninfected cells (cross-hatched bars). Data represent mean ± SEM with n = 3 mice per group. *P < 0.05 compared with PMA stimulated uninfected cells, #P < 0.05 compared with PMA stimulated RSV-infected cells.

View larger version (37K): [in this window] [in a new window]   Figure 6. Decreased oxygen radical production from SP-D–/– macrophages after RSV infection. Oxygen radical production was assessed in cells isolated from BALF 5 d after intratracheal administration of RSV. Alveolar macrophages were loaded with DCF to measure intracellular production of oxygen radicals. Alveolar macrophages from RSV-infected SP-D+/+ (solid bars) and SP-D–/– (cross-hatched bars) mice generated less oxygen radicals compared with uninfected controls (hatched and open bars, respectively). Inhibition of oxidant production was greater in alveolar macrophages from RSV-infected SP-D–/– compared with SP-D+/+ mice. Data represent mean ± SEM with n = 8 mice per group. *P < 0.05 compared with uninfected SP-D+/+ mice, #P < 0.05 compared with RSV infected SP-D+/+ mice.

View larger version (20K): [in this window] [in a new window]   Figure 7. Decreased SP-A in lungs of SP-D–/– mice after RSV infection. Concentrations of SP-A in lung homogenates were determined by Western blot analysis. SP-A concentrations were decreased in lung homogenates from SP-D–/– compared with SP-D+/+ mice 3 d after RSV infection. Each lane represents a single mouse.

View larger version (18K): [in this window] [in a new window]   Figure 8. Increased pulmonary SP-D concentrations following RSV infection. Concentrations of SP-D in BALF were determined with an ELISA. SP-D concentrations increased in the lung over 5 d after RSV infection. Five days after RSV infection, significantly greater SP-D concentrations were observed in the lungs of RSV-infected wild-type mice (solid bars) compared with uninfected wild-type mice (open bars). Data represent mean ± SEM with n = 10 mice per group, *P < 0.05 compared with uninfected wild-type mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Clearance of RSV was impaired in the lungs of SP-D^–/– mice. Although SP-D^–/– lungs have modestly enlarged airspaces, in previous studies of comparable age mice, bacteria were cleared as efficiently from SP-D^–/– as from SP-D^+/+ mice (23). Although the mechanisms underlying this abnormality are not fully clarified, defects in the innate immune response in the absence of SP-D may contribute, in part, to impaired viral clearance from the lung. SP-D is a member of the C-type lectin family of polypeptides that includes mannose-binding protein, SP-A, and conglutinin. C-type lectins share structural features, including collagenous aminoterminal and "globular" carboxyterminal domains, the latter serving as a CRD that functions in opsonization. Previously studies have shown that SP-D plays an important role in clearance of influenza virus from the lung and the clearance defect is much greater than that observed with RSV (10). These findings may reflect that the mouse is a poor host for RSV or that SP-D may have less of a role in RSV infection. However, previous studies support a role for SP-D in RSV clearance because intranasal administration of recombinant SP-D to RSV-infected mice inhibited replication of the virus in the lungs (29). In addition, SP-A levels were decreased 25% by Western blot analysis in the lungs of SP-D^–/– compared with wild-type mice (26), and in the present study, 3 d after RSV infection. Previous studies in SP-A^–/– mice demonstrated that SP-A enhanced clearance of RSV (30). It is possible that diminished SP-A levels in the lungs of SP-D^–/– mice may contribute to impaired viral clearance.

SP-D recognizes the specific RSV glycoproteins, F and G. Previous studies demonstrated that SP-D binds the highly glycosylated attachment protein G on RSV and inhibits infectivity in cultured cells (29). In the current study, SP-D bound to RSV-infected cells, and specific binding was observed for the RSV F and G glycoproteins. Binding to the F and G glycoproteins was completely inhibited with maltose, suggesting that the CRD of SP-D recognized the F and G glycoproteins. In contrast, binding of SP-D to whole RSV was only partially inhibited with maltose, suggesting that other domains of SP-D may bind to RSV-infected cells. Similar results were observed with EDTA with complete inhibition of binding of SP-D to the F and G glycoproteins and partial inhibition with whole RSV, demonstrating that the binding is calcium-dependent. In addition, competitive inhibition with F and G antibodies blocked 60% of the binding of SP-D to RSV-infected cells, suggesting that SP-D may bind other sites in addition to F and G glycoproteins on RSV-infected cells. SP-D may play an important role in RSV clearance from the lung by opsonizing the virus enhancing phagocytosis, and/or SP-D may bind RSV to prevent entry of the virus into the respiratory epithelium.

In the absence of SP-D, phagocytosis of RSV by alveolar macrophages was impaired. In vitro, SP-D enhanced phagocytosis of RSV by alveolar macrophages and neutrophils but not peritoneal macrophages, suggesting that specific receptors on phagocytic cells may be required for SP-D enhanced phagocytosis. The results support a role for SP-D in the innate immune response to RSV by binding and enhancing phagocytosis of the virus.

SP-D enhanced oxygen radical production by phagocytic cells infected with RSV. RSV inhibits the ability of macrophages and neutrophils to generate oxygen radicals (31, 32). Uninfected macrophages from SP-D^+/+ and SP-D^–/– generated similar amounts of oxygen radicals, in contrast to previous published results in which the macrophages from the SP-D^–/– mice generated greater oxygen radicals (28, 33). This difference is believed to be due to the breeding of the strain of mice (Black Swiss) over several years, consistent with findings described by Hickman and colleagues (34) that the phenotype of mice may change over several generations of breeding. In the current study, macrophages and neutrophils infected with RSV generated less oxygen radicals after PMA stimulation compared with PMA stimulation of uninfected cells. In the presence of SP-D, oxygen radical production by PMA-stimulated phagocytic cells was similar to that of uninfected cells, demonstrating that SP-D can reverse the inhibitory effects of RSV on oxidant production. Consistent with this finding, SP-D enhanced neutrophil respiratory burst responses to IAV in vitro (8). Because SP-D was added to the cells simultaneously with RSV, SP-D may bind and agglutinate the virus, enhancing uptake by the phagocytic cells resulting in enhanced oxygen radical production. However, SP-D selectively reversed the inhibitory effects of RSV on PMA-stimulated phagocytic cells, suggesting a direct effect on cells of SP-D on oxidant production.

Oxidant production by alveolar macrophages was reduced in SP-D^–/– mice after RSV infection. Whereas previous studies demonstrated increased external release of oxygen radicals by uninfected SP-D^–/– alveolar macrophages, the current study used an assay with DCF, which measures intracellular oxygen radical production. RSV is known to inhibit the respiratory burst of macrophage and neutrophils, which may contribute, in part, to increased susceptibility of the host to secondary bacterial infection. In vivo, alveolar macrophages from wild-type and SP-D^–/– mice generated less oxygen radicals after RSV infection; however, the impairment was greater in the absence of SP-D. These findings demonstrate that SP-D plays an important role in regulating macrophage oxygen radical production after viral infection.

After RSV infection, markers of inflammation, including inflammatory cells and cytokines, were increased in the lungs of SP-D^–/– mice. SP-D^–/– mice mount an immune response to RSV infection; however, the response is greatly increased compared with wild-type controls. Increased cytokine production may reflect increased cells in BALF after viral infection. Uninfected SP-D^–/– mice have increased numbers of alveolar macrophages in the lung; however, proinflammatory cytokine concentrations are not increased (33). Increased TNF-, IL-1ß, IL-6, and IFN- levels were demonstrated after IAV infection in mice wherein lymphocytic and mononuclear infiltrates predominate (35). Lung cytokine responses to IAV were similar to those observed with RSV infection in SP-D^–/– mice; however, in the absence of SP-D the neutrophilic infiltrates predominated in response to RSV.

SP-D concentrations in the lung increased after RSV infection. Although the mechanism of this observed increase is unknown, changes in SP-D synthesis or degradation may alter SP-D levels in the lung during viral infection. In previous studies, SP-D levels increased in the lungs of mice after IAV infection (10). SP-D levels were reduced in BALF from children with respiratory failure caused by viral infection (14, 36), suggesting that SP-D concentrations in the lung may vary during the course of viral infection. SP-D levels may increase in the lung early after viral infection to enhance uptake and killing by phagocytic cells. Alternatively, if viral clearance is impaired, resulting in lung inflammation, SP-D levels may decrease due to impaired production or enhanced uptake.

In summary, RSV clearance from the lung was impaired in SP-D^–/– mice and lung inflammation was more severe in SP-D^–/– mice. SP-D directly binds RSV, which may play a role in phagocytic clearance of RSV. In the absence of SP-D, oxidant production by alveolar macrophages was reduced after infection, suggesting that SP-D enhances killing of RSV and may protect phagocytic cells from the inhibitory effects of RSV on oxidant production. Because the airway is the usual portal of entry for RSV and other respiratory pathogens, the local production of SP-D is likely to play a role in innate defense responses to inhaled viruses.

	Acknowledgments

 
The authors thank William Hull for SP-D ELISA analysis and Dr. Gary Ross for Western analysis. This study was supported by HL03905 (A.M.L.), HL58795 (T.K.), HL61646, and HL56387 (J.A.W.).

Received in original form March 25, 2003

Received in final form March 5, 2004

	References

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