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Abstract
Introduction
Materials and methods
Results
Discussion
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Surfactant protein-A (SP-A) gene-targeted mice clear group B streptococcus (GBS) from the lungs at a
slower rate than wild-type mice. To determine mechanisms by which SP-A enhances pulmonary clearance
of GBS, the role of SP-A in binding and phagocytosis of GBS was assessed in SP-A (
/) mice infected
with GBS in the presence and absence of exogenous SP-A. Coadministration of GBS with exogenous
SP-A decreased GBS colony counts in lung homogenates of SP-A (/) mice. SP-A bound to GBS in a
calcium-dependent manner. Although pulmonary infiltration with macrophages was not altered in SP-A
(/) versus wild-type mice after GBS infection, the number of alveolar macrophages with phagocytosed
bacteria was lower in the SP-A (/) mice than in the wild-type mice. When SP-A was coadministered
with GBS, phagocytosis was significantly increased. Oxygen radical production by alveolar macrophages
from SP-A (/) mice infected with GBS was decreased compared with wild-type controls and was increased when SP-A (/) mice were infected in the presence of exogenous SP-A. Superoxide (SO) radical generation was deficient in macrophages from SP-A (/) mice. SP-A plays an important role in GBS
clearance in vivo, mediated in part by binding to and enhancing GBS phagocytosis and by increasing SO
production by alveolar macrophages.
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Introduction |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Surfactant protein-A (SP-A) is a 28- to 34-kD member of the collectin subgroup of the mammalian C-type lectins that also includes surfactant protein D (SP-D), mannose-binding protein, and conglutinin (1, 2). The collectins are thought to be involved in innate host defense against various bacterial and viral pathogens. For example, children deficient in mannose-binding proteins are more susceptible to bacterial infection (3). The collectins form multimeric structures resembling C1q (the first component of the complement cascade), consisting of multimeric collagenous amino-terminal domains and globular carboxy-terminal, carbohydrate binding domains (2, 4). The C-type lectins bind carbohydrate surfaces of many microorganisms mediating phagocytosis and killing by phagocytic cells (5).
SP-A is an abundant C-type lectin produced primarily by alveolar type II cells, nonciliated bronchiolar cells, and tracheobronchial gland cells in the lung. SP-A binds to specific cell-surface receptors on alveolar macrophages (9) and type II epithelial cells (10). In vitro, SP-A stimulates macrophage chemotaxis (11); enhances activity of the mannose receptor (12); and enhances the binding of serum-opsonized Staphylococcus aureus (13), non-serum-opsonized Escherichia coli, S. aureus, Hemophilus influenzae, Klebsiella pneumoniae, and Mycobacterium tuberculosis to alveolar macrophages (7, 12, 14). SP-A also binds to both the C1q receptor on monocytes and to S. aureus, promoting phagocytosis of the bacteria in vitro (17).
Alveolar macrophages are thought to play a critical role in host defense of the lung. Alveolar macrophages bind, phagocytose, and kill bacteria in association with cellular activation, release of intracellular proteases, and reactive oxygen species. Reactive oxygen species are released by activated alveolar macrophages, directly killing bacteria. In vitro, SP-A directly stimulates lucigenin-dependent chemiluminescence (13) and produces a dose-dependent enhancement of oxygen radical release from rat alveolar macrophages (18). In contrast, stimulated canine alveolar macrophages produce fewer superoxide (SO) anions in the presence of SP-A (19).
In spite of considerable in vitro evidence that SP-A is
involved in host defense, its role in vivo has only recently
been demonstrated. SP-A-deficient mice produced by
gene targeting are susceptible to group B streptococcal
(GBS) pneumonia and sepsis after intratracheal administration of the organism (20). Decreased bacterial clearance in the SP-A (/) mice was associated with decreased bacteria associated with alveolar macrophages,
but the mechanism by which SP-A contributes to bacterial
clearance in the lung remains unclear. In the present
study, we tested whether acute administration of exogenous SP-A protects SP-A (/) mice from GBS pneumonia and whether opsonization, phagocytosis, and/or oxygen radical generation were influenced by SP-A in vivo.
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Materials and Methods |
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Materials and methods
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Discussion
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Animal Husbandry
The murine SP-A gene locus was targeted by homologous
recombination as previously described. Lungs of SP-A (/)
mice do not contain detectable SP-A messenger RNA
(mRNA) or protein (21). To limit variability related to
strain differences, 129 J wild-type (+/+) and SP-A (/)
mice of the same strain were studied. Animals were
housed and studied under Institutional Animal Care and
Use Committee-approved protocols in the animal facility
of the Children's Hospital Research Foundation, Cincinnati, Ohio. Male and female mice of approximately 20 to
25 grams (35 to 42 d old) were used.
Purification of Human SP-A
Human SP-A obtained from patients with alveolar proteinosis was purified by the 1-butanol extraction method of Haagsman and colleagues (22). Purified SP-A was dissolved in 2 mg/ml Na-N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (pH 7.2) and tested with the Limulus Amoebocyte Lysate assay (Sigma, St. Louis, MO) for endotoxin. SP-A used in the present study had no detectable endotoxin (< 0.06 EU/ml).
Preparation of Bacteria
A stock culture of GBS was obtained from a clinical isolate from a newborn with systemic infection. Bacteria were
suspended in sterile phosphate-buffered saline (PBS) containing 20% glycerol and frozen in aliquots at 70°C. Bacteria from the same passage were used to minimize variations in virulence related to culture conditions. Before
each experiment, an aliquot was thawed and plated on
tryptic soy-5% defibrinated sheep blood agar, then inoculated into 4 ml of Todd-Hewitt broth (Difco Laboratories,
Detroit, MI) and grown for 14 to 16 h at 37°C with continuous shaking. The broth was centrifuged and the bacteria
were washed in PBS at pH 7.2 and resuspended in 4 ml of
the buffer. To facilitate studies, a growth curve was generated so the bacterial concentration could be determined
spectrophotometrically and confirmed by quantitative culture of the intratracheal inoculum.
Labeling of Bacteria with Fluorescein Isothiocyanate
Bacteria were harvested from agar plates 24 h after streaking, suspended in 5 ml Dulbecco's PBS (D-PBS), pH 7.2, and centrifuged for 1 min at 228 × g to remove any large
aggregates or agar. The optical density at 660 nm (OD660)
of the resulting supernatant was measured to determine
bacterial concentration. The suspension was then pelleted
at maximum speed in a microfuge and the pellet resuspended in 0.9 ml D-PBS, pH 7.2, and heated to 95°C for 10 min to kill the bacteria. The heat-killed bacteria were then
pelleted and resuspended in 1 ml 0.1 M sodium carbonate, pH 9.0. Fluorescein isothiocyanate (FITC; Molecular
Probes, Eugene, OR) was added as a 10 mg/ml stock in
dimethly sulfoxide to a final concentration of 0.01 mg/ml,
and the suspension was incubated for 1 h in the dark at
room temperature with gentle agitation. Labeled bacteria were washed 4 times for 5 min each time with D-PBS, pH
7.2, to remove unconjugated fluorophore, and finally diluted in D-PBS and stored in aliquots of 100 µl at 80°C.
Binding of SP-A to GBS
SP-A was labeled with 125I as previously described (9). Microfuge tubes were filled with 5 mM Tris and 137 mM
NaCl (Tris-buffered saline [TBS], pH 7.3), containing 0.1%
bovine serum albumin (BSA), and placed overnight at 4°C
to block possible sites of nonspecific binding to plastic.
GBS was harvested from agar plates 24 h after streaking,
and suspended in TBS (pH 7.3) containing 0.1% BSA and either 1 mM CaCl2 or 0.2 mM ethyleneglycol-bis-(
-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). The OD of
the bacterial suspension was measured at 660 nm and compared with a previously determined OD per colony-forming unit (cfu) ratio to determine bacterial number. To 200-µl
aliquots of the bacterial suspensions (2 × 107 cfu), 0 to
10,000 ng of 125I-SP-A was added. The contents were incubated for 1 h at room temperature on a rotator, and pelleted at 7,000 × g in a Beckman 12 microfuge. Pellets were
resuspended in TBS, pH 7.3, containing 0.1% BSA and either 1 mM CaCl2 or 0.2 mM EGTA; transferred to new
microfuge tubes; blocked in TBS with 0.1% BSA; and repelleted. Bacterial pellets were cut off and counted using a
-counter. Counts were expressed as counts per minute
per 1 × 107 bacteria.
Aggregation of GBS by SP-A
GBS was harvested from agar plates 24 h after streaking and suspended in 2 ml TBS, pH 7.4, containing 2 mM CaCl2. A total of 1 ml of the suspension was placed in each of two cuvettes, and the OD600 was determined using a Hitachi U-2000 Model 121-002 spectrophotometer. A total of 20 µg human SP-A or 20 µg buffer (PBS, 1 mM MgCl2, 2 mM EGTA) was added to one cuvette and the OD600 was read continuously for 4 h, interrupting the scan only to read the OD600 of the cuvette containing no protein.
Intratracheal Inoculation
GBS was administered into the respiratory tract of the mice by intratracheal inoculation of approximately 104 cfu diluted in sterile normal saline (0.9% NaCl). To deliver GBS in the presence of SP-A, GBS was diluted in 0.9% NaCl or 0.9% NaCl with 1 mM Ca2+ and SP-A in a 37°C water bath for 30 min. Because 150 and 100 µg of SP-A resulted in similar clearance of GBS from the lung, the remainder of the studies were performed with 100 µg of SP-A in saline with 1 mM Ca2+. Bacteria were delivered by intratracheal inoculation as previously described (20).
Bacterial Clearance
Quantitative cultures of lung and spleen homogenates
were performed 6, 24, and 48 h after inoculation of the animals with bacteria or bacteria with SP-A, as previously
described (20). Bacterial clearance from the lungs was determined after varying SP-A doses of 50, 100, and 150 µg.
Quantitative cultures of the lung were also performed for
heterozygous SP-A (+/) mice to determine whether a
50% reduction in SP-A would provide sufficient endogenous SP-A for bacterial clearance.
Pulmonary Pathology
Lungs were inflated via a tracheal cannula at 20 cm of pressure with 4% paraformaldehyde and removed en bloc from the thorax. Lungs were dehydrated and embedded in paraffin. Tissue sections (5 µm) were stained with hematoxylin and eosin.
Bronchoalveolar Lavage (BAL)
Lung cells were recovered by BAL. Animals were killed as described for bacterial clearance and lungs were lavaged three times with 1 ml of sterile saline. The fluid was centrifuged at 2,000 rpm for 10 min and resuspended in 1 ml PBS. Differential cell counts were performed on cytospin preparations stained with Diff-Quik (Scientific Products, McGaw Park, IN).
Clearance of Exogenous SP-A
At each time point individual mice were lavaged 3 times with 1 ml PBS at 2, 6, 24, and 48 h after intratracheal administration of 100 µg of human SP-A. SP-A in the lavage was measured using an enzyme-linked immunosorbent assay (ELISA) according to standard methodology (23). The SP-A concentrations were measured in a double-antibody ELISA, using goat and rabbit anti-SP-A antisera. SP-A concentrations were determined by comparing absorbance with a standard curve generated with purified human SP-A (5 to 100 µg/ml).
Macrophage Phagocytosis
GBS phagocytosed by alveolar macrophages in vivo were quantitated by counting FITC-labeled GBS under confocal microscopy. Lung lavage fluid was centrifuged at 1,200 rpm for 10 min; rat antimouse CD16/CD32 monoclonal antibody (Fc Block) and phycoerythrin-conjugated antimouse Mac-3 antibody (Pharmingen, San Diego, CA) were added to the cell pellet; and the pellet was incubated in the dark on ice for 30 min. The Mac-3 antibody binds to the surface of the macrophage to delineate the outline of the cell. The pellet was washed with 2 ml PBS to remove unbound antibody, and a cytospin preparation was examined by confocal microscopy by a blinded observer to assess the presence of intracellular bacteria. Serial sections through 100 random macrophages were performed to determine the percentage of macrophages with phagocytosed bacteria.
GBS phagocytosed by alveolar macrophages in vitro were quantitated by counting live FITC-labeled GBS under confocal microscopy. Eight-chamber plastic culture slides, coated with 5% poly-D-lysine, were washed twice with RPMI (Dulbecco's, containing 2.5 mg/liter gentamycin and 0.1% BSA) and used immediately. A total of 200 µl of medium containing 5 × 105 macrophages were plated and allowed to adhere for 2 h at 37°C in 5% CO2. Live FITC-labeled GBS in RPMI were opsonized with or without 25 µg/ml human SP-A for 30 min at room temperature. The medium was removed from each well and replaced with 250 µl of opsonized GBS and incubated for 45 min at 37°C in 5% CO2 at a ratio of 30 bacteria to 1 macrophage. Slides were washed 3 times with D-PBS containing 1 mM CaCl2, and extracellular fluorescent bacteria were quenched with trypan blue as described previously (24). Following two additional washes, cells were fixed with 1% paraformaldehyde in D-PBS plus 1 mM CaCl2 for 10 min, then stained with Evans blue for 2 min. The percentage of macrophages with engulfed bacteria was quantified by confocal microscopy by a blinded observer. Data are expressed as percentage increases in phagocytosis compared with phagocytosis in the absence of SP-A (control, set at 100%).
SO Anion Generation
SO anion production by alveolar macrophages was determined as described (25). At 16 h after intratracheal inoculation of GBS (104 cfu), alveolar macrophages were collected 3 times by BAL with 1 ml of dye-free RPMI media
(GIBCO, Grand Island, NY). BAL fluid (BALF) from
four mice was pooled to provide sufficient macrophages for analysis. Red blood cells in the BALF were lysed with
red cell lysis buffer (Sigma), the lavage was centrifuged at
1,200 rpm for 10 min, and the pellet was resuspended in
200 µl PBS. Differential analysis of the cells revealed
> 95% macrophages. A total of 100,000 cells was placed
in wells of a 96-well plate with 1.2 mg/ml cytochrome C,
with or without 20 µg/ml SO dismutase (SOD), in a final
volume of 200 µl of Hanks' balanced salt solution. SO anion production was determined after activation with 100 ng/ml phorbol myristate acetate (PMA). OD at 550 nm
was determined using a THERMOmax microplate reader
(Molecular Devices, Menlo Park, CA) linked to a laboratory computer. Measurements were made initially; at 5, 10, and 15 min; then every 15 min until 2 h at 37°C. OD was
converted to nanomoles of cytochrome C reduced using a
molar extinction coefficient of 21.1 mM
1cm1. Each measurement was the mean of at least two replicates with eight
determinations at each time. Data were expressed as nanomoles of cytochrome C reduced per 1 × 105 cells for total oxygen radical production. SO production was assessed
by subtracting activity in the presence of SOD from total oxygen radical production.
Statistical Methods
Because the distributions of the variables, cfu per gram of lung, cfu per gram of spleen, and percent of macrophages with bacteria were not normal, a natural log transformation was used for all analyses. Analyses of variance were performed to assess differences between the groups. Individual scores for each time point were compared using the median scores nonparametric test. Findings were considered statistically significant at probability levels < 0.05.
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Results |
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Introduction
Materials and methods
Results
Discussion
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Pulmonary Pathology after GBS Administration
To determine an appropriate bacterial dose for study, wild-type, SP-A (+/+) mice were inoculated intratracheally with GBS at concentrations of 103 to 107 cfu (4 mice/ group). The 104-cfu dose resulted in 50% mortality with the deaths occurring after 48 h at this dose. Intratracheal administration of GBS was well tolerated, and all animals survived the 48-h study period at the 104-cfu dose. No alterations in activity or physical appearance of the animals were detected throughout the 48 h of study.
No significant pathologic changes were found in the
lungs 6 h after administration of GBS to SP-A (/) and
SP-A-treated SP-A (/) mice. Mild inflammation was
observed in lungs from both groups at 24 and 48 h, consisting primarily of macrophage infiltration (data not shown).
Pulmonary infiltrates were not observed in SP-A (/)
mice inoculated with sterile 0.9% NaCl (n = 6).
Exogenous SP-A Increased Bacterial Clearance
in SP-A (/) Mice
The clearance of GBS in the SP-A (/) mice was significantly enhanced when GBS was coadministered with SP-A
(Figure 1). GBS clearance in SP-A-treated SP-A (/)
mice was similar to that in SP-A (+/+) mice. Exogenous
SP-A was equally effective in the presence and absence of
calcium. GBS clearance by SP-A (/) mice was increased by 100 and 150 µg of SP-A but not by 50 µg (Figure 2). Whereas SP-A mRNA and protein were reduced
by approximately 50% in heterozygote SP-A +/ (21)
mice, these mice cleared GBS as efficiently as wild-type
mice (Figure 3). Systemic dissemination of GBS to the
spleen occurred in 4 of 10 SP-A (/) mice and none of
10 SP-A (/) mice treated with SP-A at 24 h (data not
shown), consistent with our previous observation (20).
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Clearance of Exogenous Human SP-A
SP-A was determined in BALF after intratracheal administration of 100 µg of human SP-A. Human SP-A was reduced to 30% of starting levels 24 h after intratracheal administration and was undetectable 48 h after administration. Recovery of human SP-A was decreased when coadministered with GBS (data not shown).
Decreased Phagocytosis of Bacteria by
Macrophages in SP-A (/) Mice
BALF from SP-A (/) mice infected with GBS with or
without SP-A contained primarily alveolar macrophages
at all time points. Cell numbers and differential cell counts
of the BALF from both groups of mice were similar at 6 and 24 h (data not shown). Control experiments, in which
sterile 0.9% NaCl was injected intratracheally, showed
that the inoculation procedure did not alter the cell counts in BALF (data not shown).
125I-labeled human SP-A bound GBS in a concentration- and calcium-dependent manner in vitro (Figure 4),
but did not cause bacterial aggregation (data not shown).
Fluorescein-labeled GBS was preincubated with SP-A and
calcium before intratracheal inoculation. The method used
in this study allows detection of bacteria within the alveolar macrophage. FITC-labeled bacteria and stained macrophages were visualized under confocal microscopy with
three-dimensional imaging. Serial sections through the cell
confirmed the presence of intracellular bacteria. Phagocytosis by alveolar macrophages was decreased in SP-A (/)
mice. Coadministration of SP-A with GBS significantly increased phagocytosis but not to the level detected in alveolar macrophages from wild-type mice (Figure 5). In vitro,
SP-A enhanced phagocytosis of opsonized GBS to about
5-fold over control levels of phagocytosis by isolated alveolar macrophages. There was no detectable difference in
levels of phagocytosis by alveolar macrophages between
SP-A (+/+) and SP-A (/) mice (Table 1).
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Decreased SO Production in SP-A (/) Mice
SO production was assessed in cells isolated from BALF
16 h after intratracheal administration of GBS. More than
95% of cells in BALF from both SP-A (/) and SP-A
(+/+) mice were macrophages. Stimulation of alveolar
macrophages with a single dose of PMA produced an immediate, maximum production of oxygen radicals followed by a decay over 2 h. After stimulation with PMA,
alveolar macrophages from SP-A (/) mice produced
less total oxygen radicals and SO than did macrophages
from SP-A (+/+) mice. When SP-A (/) mice were
treated with SP-A, alveolar macrophages produced oxygen radicals at levels similar to wild-type mice. Alveolar
macrophages from SP-A-treated SP-A (/) mice were
capable of generating SO; however, the levels were lower
than in wild-type mice (Figure 6).
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Introduction
Materials and methods
Results
Discussion
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Pulmonary clearance of intratracheally administered GBS
was reduced in SP-A (/) mice compared with wild-type
mice. Coadministration of exogenous SP-A with the bacteria significantly improved bacterial clearance to that of
SP-A (+/+) mice, demonstrating an immediate reversible
defect in the SP-A (/) mouse. SP-A bound GBS in
vitro and enhanced phagocytosis in vivo. Fewer alveolar
macrophages phagocytosed bacteria in SP-A (/) mice,
and bacterial loads were consistently increased in SP-A
(/) mice. SO radical production was absent in alveolar
macrophages isolated from SP-A (/) mice. These findings support the concept that SP-A plays an important role
in the initial pulmonary host defense to GBS by binding to GBS and enhancing bacterial clearance, mediated in part
by increasing phagocytosis, opsonization, and macrophage
production of oxygen radicals.
In vitro, SP-A bound GBS in the presence of calcium, suggesting that SP-A acts as an opsonin for GBS. SP-A is a member of the C-type-lectin family of polypeptides that includes mannose-binding protein, conglutinin, and SP-D. These proteins share structural features, including collagenous amino-terminal and "globular" carboxy-terminal, carbohydrate recognition domain and all function as opsonins in vitro. In the presence of calcium, SP-A binds a variety of monosaccharides, including D-mannose, L-fucose, D-glucose, and D-galactose (2). GBS has a polysaccharide capsule consisting of repeating monosaccharides that may be recognized by the carbohydrate recognition domain of SP-A. SP-A bound to S. aureus and Streptococcus pneumoniae in vitro, and increased adherence of S. aureus to alveolar macrophages (7). Binding of SP-A to carbohydrate-dependent recognition sites on the surface of bacteria may play an important role in the early clearance of bacteria from the lungs.
The number of alveolar macrophages containing ingested bacteria was less in the SP-A (/) mice than in
wild-type mice. Coadministration of GBS with SP-A improved phagocytosis by macrophages but not to the level
of wild-type mice. The finding of increased GBS within the
macrophages of wild-type and SP-A (/) mice pretreated with SP-A, both in vitro and in vivo, supports a direct role for SP-A in bacterial phagocytosis by alveolar
macrophages. These findings are consistent with previous
studies wherein SP-A increased the phagocytosis of serum-opsonized S. aureus by alveolar macrophages (13)
and increased serum-independent phagocytosis of E. coli, Pseudomonas aeruginosa, and S. aureus in vitro (14). Ingestion of bacteria by alveolar macrophages may be important in the early elimination of some bacterial species
from the lungs (26). Studies in neonatal rabbits suggest
that immaturity of alveolar macrophages, associated with
decreased phagocytosis of GBS, increases the susceptibility to GBS infection (27). Delayed initial ingestion of GBS
by alveolar macrophages may be one factor that allows
GBS to persist and replicate in the lungs of susceptible neonates, especially premature neonates with decreased SP-A
concentrations in the lung.
Oxygen radical production by alveolar macrophages
was decreased in SP-A (/) mice. In vitro, SP-A binds to
alveolar macrophages in a concentration-dependent manner (9), mediated, at least in part, by the C1q receptors
(17). SP-A enhanced lucigenin-dependent chemiluminescence of rat alveolar macrophages (13), indicating that
SP-A may stimulate the release of SO radicals to enhance bacterial killing. However, other studies failed to demonstrate effects of SP-A on intracellular killing of bacteria by
monocytes; and SP-A-opsonized S. aureus did not induce
the production of reactive oxygen intermediates (17). Oxygen radical and SO production by alveolar macrophages
from SP-A (/) mice was decreased compared with
SP-A (+/+) mice. Pretreatment of SP-A (/) mice with
exogenous SP-A increased the total oxygen radical generation but was less effective in enhancing SO production by alveolar macrophages. The respiratory burst, although intimately connected with phagocytosis, is not
essential for killing after phagocytosis. The respiratory burst may be stimulated directly by opsonized particles
such as SP-A-bound GBS. The present study supports a
direct role for SP-A in enhancing bacterial killing through
macrophage activation and production of reactive oxygen species.
SP-A may be required to activate macrophages in vivo
so they respond to GBS infection by increasing production
of oxygen radicals. The absence of SP-A in SP-A (/)
mice may prevent normal activation of macrophages. In
the present study, we did not determine the precise mechanism whereby SP-A activates macrophages to enhance production or release of oxygen radicals. However, Kabha
and colleagues (12) demonstrated increased activity of the
mannose receptor on macrophages in the presence of SP-A.
SP-A may alter cellular receptors and enhance macrophage responses to GBS.
Bacterial burden of GBS in the lung was greater in the
SP-A (/) mice. However, coadministration of GBS
with SP-A enhanced clearance to the level of wild-type
mice. Increased clearance of GBS by exogenous SP-A was
evident between 6 and 24 h. Splenic cultures showed less
GBS in the SP-A-treated SP-A (/) mice. The recovery
of exogenous human SP-A in BALF from SP-A (/)
mice was 30% of initial concentration after 24 h and complete clearance was observed after 48 h. This clearance
time-course is consistent with previous studies with radiolabeled SP-A (28). Previously, we demonstrated that GBS
proliferated in the lungs of SP-A (/) mice. A continual
supply of SP-A may be necessary to enhance macrophage function sufficiently to prevent proliferation of GBS.
When SP-A was coadministered with GBS, calcium was
not required to enhance bacterial clearance. Because the
calcium concentration in alveolar fluid was reported to be
approximately 3.2 meq/liter (29), it is possible that endogenous calcium enhanced SP-A-dependent opsonization and phagocytosis in vivo.
During lung injury, changes in the concentration of surfactant proteins may be caused by changes in SP-A synthesis or degradation. SP-A levels are reduced in BALF from
adults with bacterial pneumonia (30) and from children
with respiratory failure (31). The concentration of SP-A
necessary for effective bacterial clearance is unknown.
Bacterial clearance in heterozygous SP-A (+/) mice was
similar to that in SP-A (+/+) mice, suggesting that a reduction of SP-A by 50% is not sufficient to increase susceptibility to bacterial infection. Bacterial clearance was
enhanced in SP-A (/) mice receiving 100 and 150 µg,
but not 50 µg, of exogenous SP-A. Thus, effective clearance of bacteria may be concentration-dependent and a
threshold concentration may be required to activate macrophages or enhance phagocytosis.
GBS frequently causes neonatal pneumonia and sepsis. Animal models of GBS pneumonia have been used to evaluate exogenous surfactant administration. Surfactant decreased bacterial growth in term rabbits. In preterm rabbits, GBS proliferated in the lungs; however, no greater than controls (32). Human amniotic surfactant, which contains small amounts of SP-A, did not improve bacterial clearance from the lungs of the rabbits (33). Concentrations of SP-A in BALF from term neonates with respiratory failure caused by GBS infection are decreased (35) compared with those of older infants, children, or adults, supporting a role for SP-A in the pathogenesis of GBS disease and supporting the concept that exogenous SP-A may be useful in prevention or treatment of neonatal GBS disease.
In summary, the present study demonstrates a role for
SP-A in pulmonary clearance of GBS in vivo. GBS clearance was associated with binding of SP-A to GBS, increased phagocytosis, opsonization, and an enhanced respiratory burst by alveolar macrophages. The airway is
usually the portal of entry for GBS and other respiratory pathogens, so local production of SP-A is likely to play a
role in innate defenses preventing pneumonia and systemic infection. Exogenous administration of SP-A enhanced clearance of GBS in SP-A (/) mice and therefore may represent a strategy for preventing or treating
pulmonary infections.
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Footnotes |
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Address correspondence to: Thomas R. Korfhagen, M.D., Ph.D., Children's Hospital Medical Center, Div. of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
(Received in original form January 13, 1998 and in revised form April 13, 1998).
Abbreviations: bronchoalveolar lavage, BAL; BAL fluid, BALF; bovine serum albumin, BSA; colony-forming unit(s), cfu; Dulbecco's PBS, D-PBS; ethyleneglycol-bis-(-aminoethyl ether)-N, N'-tetraacetic acid,
EGTA; fluorescein isothiocyanate, FITC; group B streptococcus, GBS;
optical density, OD; phosphate-buffered saline, PBS; phorbol myristate
acetate, PMA; superoxide, SO; surfactant protein-A (-D), SP-A (-D);
Tris-buffered saline, TBS.
Acknowledgments: This work was supported in part by HL28623 (T.R.K. and J.A.W.), HL58795 (T.R.K.), Cystic Fibrosis foundation, Program of Excellence Molecular Biology HL41496 and HL51134 (J.R.W.). One author (A.M.L.) is a Procter Fellow at Children's Hospital Medical Center, Cincinnati, Ohio. The authors thank Ann Maher for assistance with manuscript preparation, William Hull for SP-A analysis, and Dr. Peter Gartside for assistance with statistical methods.
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Materials and methods
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References
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1. Thiel, S., and K. Reid. 1989. Structures and functions associated with the group of mammalian lectins containing collagen-like sequences. FEBS Lett. 250: 78-84 [Medline].
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3. Super, M., J. S. Thiel, J. Lu, R. J. Levinsky, and M. W. Turner. 1989. Association of low levels of mannan-binding protein with a common defect of opsonisation. Lancet 2: 1236-1239 [Medline].
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Malhotra, B. R.,
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K. B. M. Reid, and
R. B. Sim.
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Human leukocyte C1q receptor binds other soluble proteins with collagen domains.
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