Introduction

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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, which 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 carboxyterminal, carbohydrate-binding domains (2, 4). The C-type lectins bind the 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 (AM) (9) and type II epithelial cells (10). In vitro, SP-A stimulates macrophage chemotaxis (11), directly alters macrophages by enhancing activity of the mannose receptor (12), and binds directly to the surface of some strains of bacteria, including Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli (rough), Hemophilus influenzae, Klebsiella pneumoniae (K21), and Mycobacterium tuberculosis (7, 12, 13). With some bacteria, SP-A acts as an opsonin. In addition, SP-A stimulates macrophages directly, without SP-A binding to bacteria. Direct macrophage stimulation leads to enhanced clearance of Pseudomonas aeruginosa and some strains of E. coli, K. pneumoniae, and M. tuberculosis (12, 17, 18). The binding of SP-A to carbohydrates on the surface of S. pneumoniae and S. aureus is mediated by the C-terminal lectin domain (7). SP-A also binds to both the C1q receptor on monocytes and to S. aureus, promoting phagocytosis of these bacteria in vitro (19).

P. aeruginosa is the most frequent gram-negative pathogen causing nosocomial pneumonia (20, 21). Ventilator- associated pneumonia caused by P. aeruginosa has a mortality rate of 40% to 68%, and the excess mortality appears to be related to the host-defense response to the pneumonia (22). Furthermore, septic shock and multiple organ dysfunction frequently complicate ventilator-associated pneumonia caused by P. aeruginosa (23). Mucoid P. aeruginosa is the major bacterial pathogen in cystic fibrosis lung disease, causing acute and chronic inflammation and increased morbidity and mortality. Lung injury associated with P. aeruginosa infection is related to the destructive effects of the organism on the lung parenchyma, and is exacerbated by the activation of neutrophils and other inflammatory mediators (24). Adults and children with inflammation in the lung from bacterial pneumonia (25, 26) and cystic fibrosis (27) have decreased SP-A in their bronchoalveolar lavage fluid (BALF).

During P. aeruginosa infection, both AM and polymorphonuclear leukocytes (PMN) mediate elimination of the organism. P. aeruginosa binds to the macrophage mannose receptor (28), enhancing phagocytosis and killing of the organism. Reactive oxygen species (ROS) are released by both macrophages and neutrophils, directly killing bacteria. Hypochlorous acid is an oxygen-derived reactive molecule generated through the H₂O₂-myeloperoxidase (MPO) system, with bactericidal activity (29). Oxidative killing also occurs through the production of nitric oxide (NO), which in the presence of ROS is converted to peroxynitrite, a potent bactericidal free radical (30).

Despite 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 pneumonia and sepsis after intratracheal administration of these organisms (31). The role of SP-A in pulmonary infections caused by gram-negative bacteria or other bacterial species has not been examined in vivo. In the present study, the role of SP-A in vivo in the clearance of mucoid P. aeruginosa from the lungs, phagocytosis by AM, modulation of cytokine production, and neutrophil radical generation were determined.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

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 (32). 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, OH. Male and female mice of approximately 20 to 25 g (35 to 42 d old) were used.

Preparation of Bacteria

A stock culture of a mucoid P. aeruginosa strain obtained from a clinical isolate was kindly provided by Dr. J. R. Wright (Duke University, Durham, NC). This strain was not typed in terms of the type of its lipopolysaccharide (LPS). Bacteria were suspended in 2× yeast tryptone (YT) agar with 20% glycerol, and were frozen in aliquots at 70°C. To minimize variations in virulence related to culture conditions, bacteria from the same passage were used. Before each experiment, an aliquot was thawed and plated on 2× YT agar, then inoculated into 4 ml of 2× YT broth and grown for 14 to 16 h at 37°C with continuous shaking. The broth was centrifuged and the bacteria were washed in phosphate-buffered saline (PBS) at pH 7.2 and resuspended in 4 ml of the buffer. The concentration of the inoculum was determined by quantitative culture on 2× YT agar.

Intratracheal Inoculation

Administration of P. aeruginosa into the respiratory tract of mice was accomplished by intratracheal inoculation of 10⁸ cfu diluted in sterile PBS. Mice were anesthetized with isofluorane, and an anterior midline incision was made to expose the trachea. A 30-gauge needle attached to a tuberculin syringe was inserted into the trachea, and a 100-µl inoculum of bacteria was dispersed into the lungs. The incision was closed with one drop of Nexaband. Nonpyogenic PBS was injected intratracheally as a control.

Bacterial Clearance

Quantitative cultures of lung and spleen homogenates were established at 6, 24, and 48 h after inoculation of the animals with bacteria. Mice were exsanguinated after a lethal intraperitoneal injection of sodium pentobarbital. The abdomen was opened by a midline incision, and the animal was exsanguinated by transection of the inferior vena cava to reduce pulmonary hemorrhage. The lung and spleen were removed and weighed, and each was homogenized in 2 ml of sterile PBS. Aliquots of 100 µl of homogenate and further dilutions were plated on 2× YT plates to quantitate bacteria.

Pathology

Lungs were inflated via a tracheal cannula at 20 cm of pressure with 4% paraformaldehyde, and were removed en bloc from the thorax. Lungs were dehydrated and embedded in paraffin. Each lobe of the lung was bisected, and four sections were obtained in a caudal to cranial orientation from the cut surface. Tissue sections (5 µm) were stained with hematoxylin-eosin (H&E).

Bronchoalveolar Lavage

Lung cells were recovered by bronchoalveolar lavage (BAL). Animals were killed as described for bacterial clearance, and the lungs were lavaged three times with 1 ml of sterile saline. BALF was centrifuged at 2,000 rpm for 10 min, and was resuspended in 1 ml of PBS. Differential cell counts were made on cytospin preparations stained with Diff-Quik (Scientific Products, McGaw Park, IL).

Labeling of Bacteria with Fluorescein Isothiocyanate

Bacteria were harvested from agar plates at 24 h after streaking; were suspended in 5 ml D-PBS, pH 7.2; and were centrifuged for 1 min at 228 × g to remove any large aggregates or agar. The optical density (OD) at 660 nm of the resulting supernatant was measured to determine bacterial concentration. The suspension was then pelleted at maximum speed in a microfuge, and the pellet was 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 dimethylsulfoxide (DMSO), 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 four times for 5 min each with D-PBS, pH 7.2, to remove unconjugated fluorophore, and were finally diluted in D-PBS and stored in aliquots of 100 µl at 80°C.

Macrophage Phagocytosis In Vivo

P. aeruginosa phagocytosed by AM in vivo were quantitated by scoring macrophages for intracellular FITC-labeled P. aeruginosa, using confocal microscopy. BALF was centrifuged at 1,200 rpm for 10 min, and rat monoclonal antimouse CD16/CD32 antibody (Fc Block) and phycoerythrin (PE)-conjugated Mac-3 antimouse antibody (Pharmingen, San Diego, CA) were added to the cell pellet and 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 of PBS to remove unbound antibody, and a cytospin preparation was examined with confocal microscopy to assess the presence of intracellular bacteria. Serial sections through 100 randomly chosen macrophages were examined to determine the percentage of macrophages with phagocytosed bacteria.

MPO Assay

Neutrophil accumulation in the lung was quantitated by measuring MPO activity in lung homogenates at 6 h after bacterial infection. Lungs were harvested, weighed, and homogenized in 3 ml homogenate buffer (100 mM sodium acetate, pH 6.0; 20 mM ethylenediaminetetraacetic acid [EDTA], pH 7.0; 1% hexadecyl trimethyl ammonium bromide [HETAB]). Lung homogenates were sonicated for 15 s and then centrifuged at 10,000 × g for 15 min at 4°C. The supernatants were diluted 1:15 in the homogenate buffer, and samples were pipetted as duplicates into 96-well microtiter plates (Falcon, Franklin Lakes, NJ). The samples were mixed with an equal volume of assay buffer (1 mM H₂O₂, 1% HETAB, 3.2 mM 3,3',5,5'-tetramethylbenzidine [TMB]) and the plate was read at 650 nm over a period of 4 min.

Oxygen-Radical and Superoxide-Anion Generation

Superoxide (SO)-anion production by neutrophils was determined as described (33). Sixteen hours after intratracheal inoculation of P. aeruginosa (10⁸ cfu), neutrophils were collected by BAL with 1 ml of dye-free RPMI medium (GIBCO, Grand Island, NY). Fluid from three installations was pooled. Red blood cells in the lavage fluid were lysed with red-cell lysis buffer (Sigma, St. Louis, MO), the lavage fluid was centrifuged at 1,200 rpm for 10 min, and the pellet was resuspended in 200 µl of PBS. Differential analysis of the cells revealed > 95% neutrophils. One hundred thousand cells were placed in wells of a 96-well plate with 1.2 mg/ml (~ 100 µmol/liter) cytochrome c, with or without 20 µg/ml superoxide dismutase (SOD), in a final volume of 200 µl of Hanks' balanced salt solution (HBSS). SO-anion production was determined after activation with 100 ng/ml phorbol myristate acetate (PMA). The OD at 550 nm was determined with a THERMOmax microplate reader (Molecular Devices, Menlo Park, CA) linked to a laboratory computer. Measurements were initially made every 2 min and then every 15 min for a total of 2 h at 37°C. OD was converted to nanomoles of cytochrome c reduced, using a molar extinction coefficient of 21.1 mM¹ cm¹. Each measurement was the mean of at least two replicates. Data for total oxygen-radical production were expressed as nanomoles cytochrome c reduced per 1 × 10⁵ cells. Superoxide production was assessed by subtracting activity in the presence of SOD from total oxygen-radical production.

Cytokine Production

Lung homogenates were centrifuged at 1,200 × g and the supernatants were stored at 20°C. Interleukin (IL)-6, IL-10, macrophage inflammatory protein (MIP)-2, and interferon- (IFN-) were quantitated with quantitative murine sandwich enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer's directions. Tumor necrosis factor- (TNF-) and IL-1 levels were measured with ELISAs, using goat antimurine antibody (R&D Systems) directed against TNF- or IL-1. All plates were read on a microplate reader (Molecular Devices) 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.

NO Production

NO was quantified by measuring its nitrite and nitrate oxidation products after their enzymatic conversion to nitrite. Nitrite was measured as previously described (34), by adding 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) to 100 µl of BALF. The OD at 550 nm (OD₅₅₀) was measured with a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA). Nitrite concentrations were calculated by comparison with the OD₅₅₀ of standard solutions of sodium nitrite. Nitrate concentrations were also determined. Nitrate in BALF was reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and nicotinamide adenine dinucleotide phosphate (NADPH) (160 µM) at room temperature. After 2 h, the nitrite concentration in the samples was measured with the Griess reaction.

Statistical Methods

Because the variables cfu/g of lung and cfu/g of spleen were not normally distributed, natural log transformations were used for all analyses. Analysis of variance (ANOVA) was done to assess differences between groups. Individual scores for each time point were compared with the median-scores nonparametric test. Findings were considered statistically significant at levels of P < 0.05.

	Results

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Pulmonary Pathology after P. aeruginosa Administration

To determine an appropriate bacterial dose, wild-type, SP-A^+/+ mice were inoculated intratracheally with P. aeruginosa at concentrations of 10⁴ to 10⁸ cfu (four mice/group). The wild-type mice cleared the P. aeruginosa at the lower concentrations, and experienced one death, after 48 h, at the 10⁸ concentration. Because the purpose of the study was to determine the role of SP-A in the acute phase of infection, the 10⁸ cfu dose of P. aeruginosa was chosen for study. Intratracheal administration was well tolerated, and all animals survived the 48-h postinoculation period.

Mild to moderate pulmonary infiltration was observed in SP-A^/ and wild-type mice at 6 h and 24 h after administration of bacteria. The infiltrates consisted primarily of PMN. At 48 h, inflammation persisted and was more severe in SP-A^/ than in wild-type mice (Figure 1). Four of five of the SP-A^/-mouse lungs in this experiment had consolidated alveoli, whereas none of the five wild-type lungs had consolidation at 48 h. Pulmonary infiltrates were not observed in SP-A^/ mice inoculated with sterile PBS (n = 6).

View larger version (70K): [in this window] [in a new window]   Figure 1.   Pulmonary pathology following P. aeruginosa administration. Photomicrographs of lung parenchyma in (A) SP-A/ and (B) SP-A+/+ mice were prepared 48 h after intratracheal administration of 108 cfu P. aeruginosa. Histologic sections were stained with H&E. Infiltration of PMN into the lung was more severe in SP-A/ mice. These sections are representative of five separate lungs from each genotype. (A) and (B) Magnification: ×10.

Decreased Bacterial Clearance in SP-A^/ Mice

Lung bacterial counts were significantly increased in SP-A^/ mice at 6 h and 24 h after intratracheal inoculation of P. aeruginosa (Figure 2). The difference in bacterial counts between SP-A^/ and SP-A^+/+ mice was most evident after 6 h, indicating that bacteria were cleared from the lungs of SP-A^/ mice at a slower rate than from the lungs of wild-type mice. Colony counts per lung that were greater than the initial inoculum were found in seven of 10 SP-A^/ mice as compared with two of 10 SP-A^+/+ mice at 6 h. No significant differences between bacterial counts in SP-A^/ and SP-A^+/+ mice were detected at 48 h. There was no significant difference in systemic spread of P. aeruginosa to the spleen at 6 h, 24 h, or 48 h (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 2.   Quantitation of bacteria in lung homogenates. Concentrations of P. aeruginosa were determined by quantitative culture of lung homogenates. Colony counts were significantly greater at 6 h and 24 h after administration of 108 cfu P. aeruginosa in SP-A/ (solid bar) than in SP-A+/+ (hatched bar) mice. Data are means ± SEM. *P < 0.05 versus wild-type mice.

Decreased Phagocytosis of Bacteria by Macrophages in SP-A^/ Mice

BALF from SP-A^/ mice infected with P. aeruginosa contained macrophages and PMN. At 2 h after infection, BALF from the SP-A^/ mice contained 54 ± 1.7% PMN, compared with 5 ± 4.5% for the wild-type mice (n = 4; P = 0.002). A predominately neutrophilic infiltrate was found at 6 h and 24 h in both SP-A^/ and wild-type mice. At 48 h, BALF from SP-A^/ mice contained 86 ± 4.2% PMN, compared with 60 ± 8.2% (n = 5; P = 0.048) for the wild-type mice. 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).

FITC-labeled P. aeruginosa and stained macrophages were visualized through confocal microscopy with three-dimensional imaging (Figure 3). The method used in this study allows detection of bacteria within the AM. Serial sections through the cell confirmed the presence of intracellular bacteria. Phagocytosis by AM was significantly decreased in SP-A^/ mice at 1 h after intratracheal inoculation of P. aeruginosa (Figure 4).

View larger version (51K): [in this window] [in a new window]   Figure 3.   Phagocytosis of FITC-labeled P. aeruginosa. Fluorescein-labeled P. aeruginosa were injected intratracheally into mice. One hour after infection, cytospin preparations of BALF were stained with phycoerythrin-conjugated antimouse Mac-3 antibody to delineate the perimeter of macrophages. Fluorescein-labeled P. aeruginosa and stained macrophages were visualized through confocal microscopy. The less intense staining represents binding of Mac-3 to the macrophage cell membranes. Bacteria are well delineated and stained more intensely (arrow). Serial sections through the cell confirmed the presence of intracellular bacteria.

View larger version (22K): [in this window] [in a new window]   Figure 4.   Decreased phagocytosis by AM from SP-A/ mice. Fluorescein-labeled P. aeruginosa phagocytosed by AM in vivo was quantitated in cytospin preparations of BALF through confocal microscopy. Cytospin preparations were stained with phycoerythrin-conjugated antimouse Mac-3 antibody to delineate the perimeter of macrophages. Bacteria were scored as phagocytosed only when observed within the perimeter of the cells. At 1 h, significantly fewer macrophages from SP-A/ (solid bar) mice than from SP-A+/+ (hatched bar) mice contained phagocytosed bacteria. Data represent means ± SEM. *P < 0.05 versus wild-type mice.

Increased MPO Activity in SP-A^/ Lung Homogenates

Lung MPO activity was measured to estimate total neutrophil influx into the lung. Six hours after intratracheal administration of P. aeruginosa, MPO activity was 44% greater in lung homogenates from SP-A^/ mice than in those from wild-type mice (Figure 5).

View larger version (17K): [in this window] [in a new window]   Figure 5.   MPO activity was increased in lung homogenates from SP-A/ mice. MPO activity was determined in total lung homogenates after intratracheal inoculation of 108 cfu of P. aeruginosa. MPO activity was significantly increased in homogenates from SP-A/ (solid bar) compared with SP-A+/+ (hatched bar) mice at 6 h. Data represent means ± SEM. *P < 0.05 versus wild-type mice.

Similarity of Neutrophil SO Production for SP-A^/ and Wild-Type Mice

SO production was assessed in cells isolated from BALF at 16 h after intratracheal administration of P. aeruginosa. More than 90% of cells in BALF from both SP-A^/ and SP-A^+/+ mice were PMN. Stimulation of BALF neutrophils with PMA produced a maximal level of oxygen radicals at 8 min and of SO at 60 min. Oxygen-radical and SO production by PMN were similar in cells from SP-A^/ and SP-A^+/+ mice (Figure 6).

View larger version (14K): [in this window] [in a new window]   Figure 6.   Oxygen-radical and SO production by PMN from SP-A/ mice are unaltered compared with SP-A+/+ mice. Oxygen-radical and SO production were assessed in cells isolated from BALF at 16 h after intratracheal administration of P. aeruginosa. More than 90% of cells in BALF from both SP-A/ and SP-A+/+ mice were PMN. The reduction of cytochrome c in the presence (B) and absence (A) of SOD was measured for PMA-stimulated neutrophils. Data are expressed as nanomoles of cytochrome c reduced per 1 × 105 cells for total oxygen-radical production (A), and with SOD for SO production (B). There was no difference in oxygen-radical and SO production between PMN from SP-A/ (solid circle) and SP-A+/+ (solid square) mice. Data are means of eight determinations at each time point.

Cytokine Levels in Lung Homogenates

Infection with P. aeruginosa significantly increased levels of the proinflammatory cytokines TNF-, IL-1, IL-6, and MIP-2 in lung homogenates from SP-A^/ and wild-type mice. At 2 h after infection, but not at 6 h or 24 h, levels of TNF-, IL-6, and MIP-2 were significantly higher in lung homogenates from SP-A^/ mice (Figure 7). Concentrations of the antiinflammatory cytokines IL-10 and IFN- were relatively low in lung homogenates from SP-A^/ and wild-type mice. At 24 h after infection, IL-10 concentrations from lung homogenates of SP-A^/ mice were greater than in those from wild-type controls.

View larger version (22K): [in this window] [in a new window]   Figure 7.   Cytokine levels in lung homogenates. TNF-, IL-1, IL-6, IL-10, MIP-2, and IFN- were assessed in lung homogenates from SP-A/ (solid bar) and SP-A+/+ (hatched bar) mice. Increased concentrations of the proinflammatory cytokines TNF-, IL-6, and MIP-2 were found in lung homogenates from the SP-A/ mice at 2 h. Increased concentrations of IL-10 and IFN- (antiinflammatory cytokines) were detected in lung homogenates from SP-A/ compared with wild-type mice at 2 h, but the concentrations were relatively low. Data are expressed in pg/ml, and represent means ± SEM values with n = 10 mice per group. *P < 0.05 versus wild-type mice.

Increased Nitrite in BALF from SP-A^/ Mice

NO production after P. aeruginosa infection was measured as nitrite in BALF. At 6 h after P. aeruginosa infection, SP-A^/ and wild-type mice showed no difference in nitrite production. At 24 and 48 h, increased nitrite levels were found in BALF from the SP-A^/ mice (Figure 8). To examine nitrate production, nitrate reductase was used to convert nitrate to nitrite. There was no difference in nitrate production between the SP-A^/ and wild-type mice at 6, 24, or 48 h.

View larger version (26K): [in this window] [in a new window]   Figure 8.   Increased nitrite concentrations in BALF from SP-A/ mice following infection. Nitrite in the BALF was measured with the Griess reaction. Nitrate was reduced to nitrite with nitrate reductase, and the total nitrite/nitrate production was measured with the Griess reaction. At 24 and 48 h after intratracheal inoculation of 108 cfu P. aeruginosa, nitrite levels in BALF from SP-A/ mice (solid bar) were increased over those in BALF from SP-A+/+ mice (hatched bar). Data represent means ± SEM. *P < 0.05 versus wild-type mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Pulmonary clearance of intratracheally administered mucoid P. aeruginosa was reduced in SP-A^/ mice. Lung inflammation and bacterial burden were increased, and fewer organisms were phagocytosed by AM in vivo in the absence of SP-A. These findings support the concept that SP-A plays an important role in the initial pulmonary host defense against mucoid P. aeruginosa.

The number of bacteria phagocytosed by AM was smaller in the SP-A^/ than in the wild-type mice. These findings are consistent with those of previous in vitro studies wherein SP-A increased the phagocytosis of serum- opsonized S. aureus by AM (13), and increased serum-independent phagocytosis of E. coli, P. aeruginosa, and S. aureus (14). Ingestion of bacteria by AM may be important in the early elimination of some bacterial species from the lungs (35), and SP-A can enhance phagocytosis by macrophages without acting as an opsonin (12, 14). SP-A does not bind directly to P. aeruginosa, but activation of macrophages by SP-A may be critical to macrophage phagocytosis of mucoid Pseudomonas. Delayed initial ingestion of P. aeruginosa by AM may be one factor that allows P. aeruginosa infection to become established in the lung. Intratracheal administration of SP-A increased phagocytosis of group B streptococci (GBS) in SP-A^/ mice, suggesting that SP-A plays an immediate and direct role in the clearance of GBS by binding directly to these organisms and acting as an opsonin (36). In contrast, preincubation of SP-A with the mucoid Pseudomonas used in this study did not enhance clearance at 6 h or 24 h after infection. SP-A is not an opsonin for mucoid P. aeruginosa, and it may be necessary to pretreat lungs with SP-A to activate macrophages for nonopsonic bacterial clearance.

Decreased pulmonary clearance of P. aeruginosa was detected at 6 h and 24 h, but not at 48 h after infection. In previous studies with GBS, significant differences in lung bacterial burden were detected at 6, 24, and 48 h (31). With GBS, the initial inoculum was 10⁴ bacteria. Colony counts per lung that were greater than the initial inoculum were found in six of 10 SP-A^/ compared with none of 10 SP-A^+/+ mice at 6 h, with colony counts of 10⁵ to 10⁶ cfu in the lung (31). In the present study, P. aeruginosa proliferated in some of the SP-A^/ and wild-type lungs at 6 h, but not to the same extent. P. aeruginosa counts were about 2-fold greater in four of 10 and 4-fold greater in three of 10 SP-A^/ mice. In the two wild-type mice in which P. aeruginosa proliferated in the lung, counts were also 2- to 3-fold greater.

Infiltration of neutrophils following administration of bacteria was more intense in SP-A^/ than in SP-A^+/+ mice. At 1 and 2 h after P. aeruginosa infection, BALF from SP-A^/ mice contained a higher percentage of neutrophils than that from wild-type mice. Enhanced neutrophil chemotaxis may have resulted from the increased cytokine production in the lungs of the SP-A^/ mice. The role of SP-A in modulating cytokine production is controversial. SP-A has been found to stimulate TNF-, IL-1, and IL-6 production by mononuclear cells (37), and TNF- production by macrophages and THP-1 cells (38). In contrast, McIntosh and Wright (39) reported that SP-A blunted TNF- release from LPS-stimulated macrophages. The finding in the present study that cytokine production was more modest in SP-A^+/+ than in SP-A^/ mice in vivo supports the McIntosh and Wright study suggesting that SP-A decreases the release of cytokines in response to bacterial infection. It is unclear from the current study whether these differences are directly related to the absence of SP-A or to the severity of infection and failure of early bacterial clearance.

SP-A^/ mice had more severe inflammation in their lungs than did SP-A^+/+ mice at 48 h after infection, despite clearance of P. aeruginosa. Greater concentrations of IL-10 and IFN- (antiinflammatory cytokines) were detected in lung homogenates from SP-A^/ than from wild-type mice early after infection, but the concentrations were relatively low compared with levels usually detected at later time points. Previous studies showed that neutralization of IL-10 increased mortality with LPS (40), and that treatment with IL-10, in the presence of IFN-, decreased lung injury and mortality from P. aeruginosa (41). In the present study, 24 h after infection, both IL-10 concentrations in lung homogenates from SP-A^/ mice and lung inflammation were greater in SP-A^/ mice. However, in the SP-A^/ model in the present study, the observed increase in IL-10 was not sufficient to modify the inflammatory response.

MPO activity in the lung and increased nitrite in the BALF of SP-A^/ mice were observed after P. aeruginosa infection. The increased MPO activity in lungs from SP-A^/ mice is consistent with increased neutrophilic infiltration after infection. MPO and NO may play roles in host defense by contributing to bacterial killing. NO reacts with SO to form peroxynitrite, which is a potent bactericidal radical (30). SP-A enhances NO production by isolated AM in vitro (42). The current study demonstrated increased nitrite concentrations in BALF from SP-A^/ mice at 24 and 48 h after P. aeruginosa infection, suggesting that nitrite production was not a critical determinant in the early clearance of P. aeruginosa in this model.

Oxygen-radical and SO production by neutrophils were not different in SP-A^/ and wild-type mice. Macrophages are the primary phagocytic cells resident in the lung. During infection, neutrophils enter the lung from the circulation. In vitro, lung surfactant has been found to inhibit the neutrophil respiratory burst (43), although SP-A alone had no effect on the neutrophil lucigenin-dependent chemiluminescence response (13). In the present study, no deficit in oxygen-radical production by neutrophils was observed in SP-A^/ mice, suggesting that other mechanisms account for the impaired clearance of P. aeruginosa seen at the early time points after infection.

Mucoid P. aeruginosa was studied as a model for evaluating the in vivo role of SP-A in host immunity against gram-negative organisms. SP-A does not bind directly P. aeruginosa in vitro (14). In contrast, in vitro, SP-A binds E. coli with a rough LPS phenotype (LPS deficient in O polysaccharide and fragments of the core oligosaccharide), but not E. coli with a smooth LPS phenotype (44). Strain differences in P. aeruginosa may also result in variable clearance and pulmonary inflammation in vivo, even though in the absence of SP-A, mucoid P. aeruginosa is cleared by 48 h, showing that other components of alveolar lavage or inflammatory cells contribute to killing of the organism in vivo. Other factors may include complement, lysozyme, lactoferrin, antibodies, and defensins. SP-A acts in the early phase of infection, as part of the innate immunity against mucoid P. aeruginosa infection.

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 (25) and from children with respiratory failure (26). The concentration of SP-A necessary for effective bacterial clearance is unknown. Transgenic mice overexpressing IL-4 in the lung have increased concentrations of SP-A in the lung, and demonstrate enhanced pulmonary clearance of P. aeruginosa (45). Previous studies of group B streptococcal pneumonia demonstrated that exogenous administration of SP-A improved pulmonary clearance of the bacteria (36). The present study suggests that SP-A deficiency is associated with susceptibility to infection with both GBS and mucoid Pseudomonas.

In summary, the present study demonstrates the role of SP-A in pulmonary clearance of P. aeruginosa in vivo. P. aeruginosa causes pulmonary infections with mechanical ventilation, cystic fibrosis, or malignancies, conditions that may also be associated with decreased concentrations or activity of SP-A in the lung. Because SP-A interacts with common respiratory pathogens, including S. aureus, S. pneumoniae, group B streptococcus, H. influenzae, E. coli, and P. aeruginosa, lack of SP-A may limit pulmonary host defense in both pediatric and adult patients.