Introduction

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The collectin family of proteins, which includes pulmonary surfactant protein (SP)-A and SP-D, appears to be involved in innate (e.g., nonantibody) mediated immunity (reviewed in [1]). The collectins, so named because they contain a collagen-like domain and a calcium-dependent lectin domain, stimulate a variety of immune cell functions, including phagocytosis and chemotaxis.

The pulmonary collectins SP-A and SP-D are synthesized by both alveolar type II cells, which are the site of synthesis of surfactant lipids and the other SPs, SP-B and SP-C, and the nonciliated bronchiolar cells, which also synthesize SP-B but do not produce SP-C (2, 3). Although both SP-A and SP-D have been named "surfactant proteins," only SP-A has been shown to facilitate the SP-B- mediated surface tension-reducing properties of surfactant lipids (4) and has been shown to be involved in the organization of surfactant lipids into the extracellular lattice-like form of surfactant known as tubular myelin. The functional significance of these in vitro observations is unclear inasmuch as mice made SP-A-deficient by homologous recombination have only minimal perturbations in their surfactant function and surfactant pool size (5). SP-D has not yet been demonstrated to participate in any structural organization or surface tension-reducing functions of surfactant.

Both SP-A and SP-D have been shown to be involved in pulmonary host defense, although the role of SP-A has been studied to a far greater extent. For example, SP-A has been found to modulate such immune cell functions as oxygen radical production (6, 7) and chemotaxis (8) of alveolar macrophages. The adherence of Pneumocystis carinii (9) and Mycobacterium tuberculosis (10, 11) to alveolar macrophages is increased by SP-A. In vitro studies have demonstrated that SP-A increases the phagocytosis by alveolar macrophages of viruses (12, 13) and bacteria such as Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenza (14). Recently, LeVine and coworkers reported that SP-A-deficient mice, generated by gene ablation, cleared intratracheally instilled group B streptococcus (19) and P. aeruginosa (20) more slowly than did wild-type mice. Recent studies with SP-D gene- targeted mice showed that an SP-D deficiency results in an increased alveolar surfactant pool (21, 22), suggesting that SP-D contributes to surfactant homeostasis.

SP-D has been shown to regulate the function of a variety of immune cells. For example, SP-D binds directly to alveolar macrophages (23, 24) and blood leukocytes (25), and thus, apparently enhances oxygen radical production (26). SP-D stimulates the migration of human neutrophils, monocytes, and macrophages in vitro (25, 27, 28). In addition, SP-D has been found to aggregate influenza A virus, inhibit viral hemagglutination activity (29), and enhance neutrophil binding of influenza virus (12). SP-D binds and aggregates other organisms, including gram-negative bacteria such as Escherichia coli (16, 30) and Klebsiella pneumoniae (31), as well as P. carinii (32), Aspergillus fumigatus conidia (27), and Cryptococcus neoformans (33). The binding and aggregation of gram-negative bacteria appears to be at least partially mediated by binding to the bacterial lipopolysaccharide (31). Although SP-D binds to numerous organisms, it has been demonstrated that it does not enhance either the phagocytosis of E. coli serotype J5 by alveolar macrophages or influenza virus A by neutrophils (12, 34). In contrast, SP-D does enhance the uptake of A. fumigatus conidia (27), E. coli, S. pneumoniae, and S. aureus by neutrophils (35).

Pseudomonas aeruginosa is an important pathogen of the respiratory tract and is a significant cause of nosocomial pneumonia, ventilator-associated pneumonia, and pneumonia in immunocompromised patients (reviewed in [36]). P. aeruginosa pneumonia is associated with a higher mortality rate in these situations. In addition, several P. aeruginosa strains colonize and proliferate in the airways of patients with cystic fibrosis (CF) (reviewed in [37]). The goal of the current study was to investigate the role of SP-D in the clearance of P. aeruginosa by alveolar macrophages.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents and Media

Fluorescein isothiocyanate (FITC) was obtained from Molecular Probes (Eugene, OR). Dulbecco's phosphate-buffered saline (D-PBS), RPMI 1640, and gentamicin were obtained from GIBCO BRL (Grand Island, NY). All other chemicals, except as noted, were obtained from Sigma Chemical Company (St. Louis, MO). Bovine serum albumin (BSA; Sigma Chemical) used in the phagocytosis buffer was from fraction V, fatty acid-free, cell-culture tested, and reported to have endotoxin levels of < 0.1 ng/mg.

Purification of SP-A and SP-D

SP-A was purified from human alveolar proteinosis patients as previously described (38). Briefly, SP-A was extracted from lavage fluid by butanol extraction, and sequential solubilization in octylglucoside and 5 mM Tris, pH 7.4. The SP-A was treated as previously described with polymyxin B agarose beads to remove endotoxin (38). SP-A preparations were found to have < 0.1 pg/µg of endotoxin by the Limulus amebocyte lysate assay QCL-1000 (BioWhittaker, Walkersville, MD).

Rat SP-D was purified from the lavage of rats injected with silica to augment surfactant content (39). Approximately 30 d after the silica instillation, the lungs were lavaged and the lavage fluid was centrifuged at approximately 27,000 × g_av for 30 min at 10°C. The supernatant was mixed with maltose-sepharose prepared as described by Fornstedt and Porath (40) and incubated overnight at 4°C. The beads were washed by centrifugation a total of seven times with 50 mM Tris (pH 7.4), 150 mM NaCl, and 5 mM CaCl₂; and calcium-dependent maltose binding proteins were then eluted with 50 mM Tris (pH 7.4), 150 mM NaCl, and 10 mM ethylenediaminetetraacetic acid (EDTA). SP-D was further purified by gel filtration chromatography as described by Persson and associates (41). Purified SP-D was stored at 4°C in either 2 or 10 mM EDTA. The SP-D solution was recalcified before its use in phagocytosis assays. Although some of these studies were done before we had established routine methods for analyzing the endotoxin content of SP-D, four of the six preparations used were analyzed for endotoxin and found to contain an average of 1.2 ± 0.9 pg/µg SP-D (mean ± standard error of the mean [SEM] for four preparations, range 0.06 to 3.8 pg/µg SP-D).

Recombinant rat SP-D was purified from the medium of Chinese hamster ovary cells that had been stably transfected with the SP-D complementary DNA (cDNA) ligated into the pEE14 vector (Celltech Therapeutics, Ltd., Berkshire, UK) as previously described (42). The recombinant protein was radiolabeled by inclusion of ³H-proline in the incubation medium and was purified by maltose affinity chromatography (42). The specific activity was approximately 6 × 10⁴ cpm/µg. The SP-D cDNA was a kind gift from Dr. James Fisher, University of Colorado, Denver, CO.

Bacteria

One mucoid strain of P. aeruginosa (S470) was a clinical isolate from a patient with CF at the University of North Carolina-Chapel Hill Medical Center and was a generous gift from Dr. Roy Hopfer (Medical Microbiology Laboratory, UNC-CH Medical Center, Chapel Hill, NC). Two additional mucoid and two nonmucoid clinical isolates were obtained from the Duke University Clinical Microbiology Laboratory (Durham, NC). A nonmucoid P. aeruginosa strain (No. 27853) and H. influenzae strain (No. 9006) were from American Tissue Culture Collection (Rockville, MD). Pseudomonas strains were grown on nutrient agar (Difco, Detroit, MI) with 20% horse serum. H. influenzae was cultured on GCII Agar plates (Becton-Dickinson, Cockeysville, MD). The colony-forming units (CFU)/ml for each strain were titrated for an optical density (OD) of 660 nm.

Labeling of Bacteria with FITC

Overnight cultures of the bacteria were collected, resuspended in D-PBS (pH 7.2), and heated in a water bath at 95°C for 1 h when heat-killed bacteria were utilized. Subsequently, the tubes were centrifuged at approximately 12,000 × g. The bacteria were resuspended in 1 ml of 0.1 M sodium carbonate buffer, pH 9.0. FITC, at 10 mg/ml in dimethyl sulfoxide, was added to a final concentration of 0.1 mg/ml. The bacteria were incubated for 1 h at room temperature with continuous shaking while protected from direct light. The bacteria were subsequently centrifuged and washed with D-PBS three times or until the supernatant had cleared. The fluorescently labeled bacteria were stored at 80°C.

Isolation of Alveolar Macrophages

Adult male Sprague-Dawley rats (Charles River Laboratories, Raleigh, NC) were killed by pentobarbital overdose. The tracheas were cannulated and the lungs were removed. The lungs were lavaged to total lung capacity six times with a buffer containing 140 mM NaCl, 6 mM glucose, 2.5 mM phosphate buffer, 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes), and 0.2 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), pH 7.4. Subsequently, the lungs were lavaged twice with the same buffer containing 2 mM CaCl₂ and 1.3 mM MgSO₄ instead of EGTA. The lavage was centrifuged at 188 × g, at 25°C for 10 min. The supernatant was discarded and the cells were resuspended in RPMI 1640 with gentamicin 0.01 mg/ml at 1 × 10⁶ cells/ml. LabTek eight-chamber glass slides (Nalge Nunc, Inc., Naperville, IL) were precoated with poly-D-lysine (0.02 mg/ ml) as previously described (43). A total of 2 × 10⁵ cells were plated per chamber and allowed to adhere for 2 h at 37°C in 5% CO₂, except as indicated in the later section that describes the electron-microscopic studies. Cytospins of lavages stained with Hemacolor stain kit revealed macrophage populations greater than 95% of total cells.

Phagocytosis Assay for Fluorescence Microscopy

SP-A (25 µg/ml) and SP-D at various concentrations were aliquotted into microfuge tubes and diluted with D-PBS. FITC-labeled bacteria were added to the samples at a final ratio of approximately 100 bacteria per macrophage. The bacteria-protein mixtures were allowed to incubate for 15 min and were relabeled to blind the samples. The medium from the alveolar macrophages was aspirated and replaced with 0.2 ml of D-PBS with 1 mM CaCl₂ and 0.1% BSA. Subsequently, 0.05 ml of the bacteria-protein mixture was added to each chamber. The slides were centrifuged at approximately 25 × g for 3 min in a Jouan B3.11 tabletop centrifuge with microtiter plate-holders and then placed at 37°C, 5% CO₂ for 1 h. The phagocytosis was terminated with cold PBS. The chambers were removed, unbound bacteria was removed by washing with PBS, and ethidium bromide was added at 20 µg/ml to quench the extracellular fluorescence. The samples were analyzed in a blinded fashion at a magnification of ×2,000 with an epifluorescence microscope. The phagocytosis index was defined as the number of cells containing bacteria as a percentage of the number of cells that contained bacteria with no added protein (control).

For some studies, 2.5 × 10⁵ alveolar macrophages in 0.2 ml RPMI were adhered to LabTek eight-chamber plastic slides precoated with lysine or SP-D (25 µg/ml), for 2 h at 37°C in 5% CO₂. FITC-labeled P. aeruginosa, 1 × 10⁶, were incubated at 37°C with SP-D (5 µg/ml) or buffer alone in 0.2 ml D-PBS containing 1.0 mM CaCl₂ and 0.1% BSA, pH 7.4, for 1 h at 37°C with rotation. The medium from the alveolar macrophages was removed and replaced by the bacteria-protein mixture and incubated at 37°C in 5% CO₂ for an additional hour. Phagocytosis was terminated by washing the adhered macrophages with cold PBS. Extracellular fluorescence was quenched by addition of trypan blue before fixation in 1% paraformaldehyde and staining of the macrophages with Evans blue. Slides were mounted with coverslips with 1,4-diazabicyclo(2.2.2.) octane (DABCO; Kodak, Rochester, NY), dissolved as a 25 mg/ml solution in 90% glycerol, 0.27 mM KCl, 0.15 mM KH₂PO₄, 13.7 mM NaCl, and 0.81 mM Na₂HPO₄, pH 8.6 (DABCO) as previously described (43). The samples, which were blinded to the observer, were viewed with an epifluorescence microscope at ×400 magnification. Random macrophages, 100 to 200, were viewed for the presence of fluorescent particles, and the percent of macrophages containing fluorescent particles was determined for each sample.

For opsonization studies, 5 µg/ml SP-D was preincubated with 10⁷ P. aeruginosa for 60 min at 37°C. Control bacteria were incubated without SP-D. The bacteria were washed by centrifugation, resuspended in RPMI, and added to macrophages adhered to Lab-Tek chamber slides as described earlier. After 2 h incubation at 37°C, the cells were washed, extracellular fluorescence was quenched with trypan blue (0.1% for 15 min), and cells were fixed with paraformaldehyde and mounted. Phagocytosis was determined by microscopic analysis of 100 to 200 random macrophages for the presence of fluorescent particles.

Phagocytosis Assay for Fluorescence-Activated Cell Sorter Analysis

Phagocytosis was assessed using methods described by van Iwaarden and colleagues (13) and Van Strijp and associates (44) with minor modifications. Alveolar macrophages were diluted to 5 × 10⁵ cells/0.25 ml of PBS containing 0.9 mM CaCl₂ and 0.1% BSA and dispensed into microfuge tubes precoated for 1 h at 4°C with 1% BSA. SP-D of varying concentrations and fluorescently labeled bacteria were added at a ratio of 100:1 (bacteria:macrophages). Incubation was continued at 37°C for 1 h in a shaking incubator. Phagocytosis was stopped with cold PBS and the cells were washed three times and divided into two tubes, one of which was immediately fixed with 1% formaldehyde. The second tube of cells was treated with 0.3 ml of trypan blue (0.2 mg/ml) to quench extracellular fluorescence, centrifuged, and washed twice with PBS. The mean channel fluorescence was calculated from the observed frequency distribution as measured by fluorescence-activated cell sorter (FACS) of approximately 10,000 cells/sample, and phagocytosis was expressed as a percent increase over control (no added SP-D).

Phagocytosis Assay for Electron Microscopy

Alveolar macrophages (1 × 10⁶ cells/ml) were plated at a final density of 3 to 3.5 × 10⁶ cells/chamber on LabTek single-chamber slides that had been precoated with poly-D-lysine. Microfuge tubes were prepared with SP-A (25 µg/ ml), SP-D at various concentrations, and bacteria (at either 100:1 or 500:1 final ratios), and incubated for 15 min. After replacing the D-PBS media with 1 mM CaCl₂, the surfactant protein-bacteria mixtures were added to the cells in final total volume of 4 ml. The slides were centrifuged as previously described and incubated for 1 h at 37°C and 5% CO₂. The phagocytosis was terminated with 2% glutaraldehyde/paraformaldehyde (0.085 M sodium cacodylate buffer). The cells were scraped from the slides and centrifuged at 2,500 × g for 8 min. The pellets were postfixed in 2% osmium tetroxide, stained with 2% uranyl acetate, dehydrated in a graded series of acetone, and then transferred and embedded in Poly/Bed 812 Resin (Polysciences, Inc., Warrington, PA). Thin sections were cut using a diamond knife and placed on nickel-coated grids for electron-microscopic studies. The samples were analyzed with a JEOL 1200 electron microscope. The grids were scored in a random manner by a microscopist who was blinded to the samples. The number of cells with intracellular bacteria and the total number of intracellular bacteria per cell were counted. In the event that the bacteria were fragmented in the micrograph section, only those profiles that still possessed more than half of the circumference of the bacterial cell were counted. The data were expressed as a percentage of the control (no protein added), assuming the amount of phagocytosis for this group to be 100%.

Bactericidal Assay

The bactericidal effects of SP-D were assayed according to minor modifications of the method described by Pikaar and coworkers (16). Alveolar macrophages were diluted to a final concentration of 2.5 × 10⁶/ml in a buffer of 140 mM NaCl, 5 mM KCl, 2.5 mM Na₂PO₄, 10 mM Hepes, 2 mM MgSO₄, 2 mM glucose, 2 mM CaCl₂, and 0.1% BSA. P. aeruginosa (approximately 300,000 to 500,000) were mixed with SP-D and added to 500,000 macrophages in 0.2 ml of buffer. After 60 min incubation at 37°C with agitation, the cells were lysed by addition of 1.8 ml of ice-cold water. Aliquots (0.01 ml) of 10-fold serial dilutions were plated on Nutrient Agar plates containing 10% horse serum and incubated at 37°C for 18 h, after which time the number of recovered CFUs was quantitated.

Aggregation Assay

The aggregation of bacteria by SP-A and SP-D was studied as previously described (43). Bacteria were diluted to a final OD at 700 nm (OD_700nm) of approximately 0.5 to 0.7 (equivalent to 1.8 × 10¹⁰ bacteria/ml for S470, 7 × 10⁸ bacteria/ml for 27853, and 7 × 10⁸ bacteria/ml for E. coli) in PBS containing 0.9 mM calcium. The OD_700nm was measured at intervals over 2 h in the presence or absence of either SP-A (25 µg/ml) or SP-D (5 µg/ml) using a Hitachi U-2000 model 121-002 spectrophotometer. For some studies, the samples were examined by fluorescence microscopy to determine whether microscopic aggregation was occurring.

Binding of ³H-SP-D to Bacteria

Microfuge tubes were preincubated with PBS containing 0.9 mM CaCl₂ and 1% BSA at 4°C overnight to block possible nonspecific binding sites. Bacteria were scraped from agar plates and resuspended into PBS containing 0.9 mM CaCl₂ and 0.1% BSA. The OD_660nm of the bacterial suspensions was measured and the number of bacteria was calculated on the basis of the previously determined extinction coefficient for each strain of bacteria. Bacteria (4 × 10⁹) in 0.5 ml of PBS containing 0.1% BSA and either 0.9 mM CaCl₂ or 7 mM EDTA were added to microfuge tubes with varying amounts of ³H-SP-D. The tubes were incubated at room temperature with gentle shaking for 30 min, and then centrifuged at 6,000 × g for 5 min to sediment the bacteria. The bacteria were washed once with PBS containing either calcium or EDTA, transferred to new tubes, and washed one more time. The bacteria were finally resuspended into 0.1 ml of PBS containing 0.1% BSA, transferred to scintillation vials containing 4 ml scintillation cocktail (Ecolite; ICN, Costa Mesa, CA), and analyzed for radioactivity in an LS 1800 scintillation counter (Beckman, Fullerton, CA).

Statistical Analysis

When the response of the cells was expressed as a percent control (usually no SP-D or SP-A), the data were analyzed using a multiplicative model that results in a value . This value relates the control value P_O to the treatment value P_O which, when multiplied by 100, is equivalent to the percentage control response. This value  was analyzed using Student-Newman-Keuls t test with the null hypothesis that  = 1 (or 100% control). For data that were not normalized to control, a two-sided, two-tailed t test was used for the analysis.

	Results

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Phagocytosis of P. aeruginosa by Alveolar Macrophages

Initially, the effects of SP-D on phagocytosis of heat-killed mucoid P. aeruginosa and H. influenzae were characterized. As shown in Table 1, SP-D at 5 µg/ml enhanced the macrophage uptake of heat-killed P. aeruginosa approximately 2-fold as assessed by both fluorescent and electron microscopy. Electron microscopy (Figure 1) revealed the presence of intracellular bacteria, some within phagocytic vacuoles. Bacteria were also seen surrounded by pseudopods and in close proximity to the macrophage membrane. From the electron-microscopic analysis of two individual experiments it was determined that, in addition to increasing the percentage of macrophages containing bacteria, SP-D also increased the number of bacteria per cell by averages of 163 and 224%.

View larger version (134K): [in this window] [in a new window]   Figure 1.   Electron microscopic analysis of phagocytosis of heat-killed mucoid P. aeruginosa. Mucoid P. aeruginosa was killed by heat treatment and incubated with alveolar macrophages adhered to lysine-coated LabTek chamber slides. After 60 min of incubation, unbound bacteria were removed by washing and the cells were treated with glutaraldehyde and paraformaldehyde, scraped, postfixed in osmium, stained with uranyl acetate, dehydrated in a graded series of acetone, and embedded in Poly/Bed 812 resin. Arrowheads indicate ingested organisms, one of which is in a secondary lysosome and is partially degraded. Original magnification: ×8,500.

SP-D only moderately increased the uptake of H. influenzae as assessed by fluorescence microscopy and had no significant effect on uptake as assessed by electron microscopy (Table 1). In contrast, SP-A had no effect on the uptake of heat-killed P. aeruginosa but significantly increased the uptake of H. influenzae, consistent with our previous observations (43).

The effects of SP-D on stimulation of macrophage phagocytosis of heat-killed P. aeruginosa were concentration- dependent (Figure 2) and reached an apparent maximum increase of approximately 300% at a concentration of SP-D of 1 µg/ml. Previous studies have shown that the effects of SP-D on immune cell function are biphasic with maximal responses induced by concentrations in the ng/ml range (25). However, the effects of 50 and 100 ng SP-D/ml on phagocytosis were less than that observed with 500 ng/ ml (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2.   Concentration-dependent effects of SP-D on phagocytosis of heat-killed mucoid P. aeruginosa. Fluorescently labeled P. aeruginosa were incubated with various concentrations of SP-D for 15 min before addition to alveolar macrophages adhered to lysine-coated slides. After 60 min of incubation, unbound bacteria were removed by washing and the fluorescence of extracellular bacteria was quenched with ethidium bromide. Samples, which were previously coded so that the analysis was blinded, were viewed by epifluorescence microscopy. The percentage of cells that had phagocytosed bacteria in the absence of SP-D (control) was 11.22 ± 0.5%. *Significantly different from control. Values shown are means ± SEM for n = 3-7 experiments, except for the value at 0.01 µg SP-D/ml, which is the average of two experiments.

To investigate the effects of SP-D on uptake of live P. aeruginosa, two strains of P. aeruginosa, one mucoid and one nonmucoid, were fluorescently labeled and phagocytosis was analyzed by light microscopy. Opsonization of live mucoid P. aeruginosa with SP-D significantly increased its uptake by alveolar macrophages. Electron-microscopic analysis confirmed the light-microscopic analysis (Table 2). Quantitative analysis of one electron-microscopic study showed that SP-D increased the number of bacteria per cell by 124% for the live nonmucoid strain and 224% for the live mucoid strain. As shown in Figure 3, electron-microscopic images showed that several P. aeruginosa are within the cell, and a few extracellular bacteria very close to the macrophage surface can also be seen.

View larger version (152K): [in this window] [in a new window]   Figure 3.   Electron microscopic analysis of phagocytosis of live nonmucoid P. aeruginosa. Samples were processed as described in the legend of Figure 1. Multiple intracellular organisms can be seen. Arrowheads indicate ingested P. aeruginosa in a section that is almost longitudinal. Original magnification: ×7,500.

FACS analysis was used to determine whether SP-D enhanced the uptake of other strains of P. aeruginosa. For these studies, fluorescence due to extracellular bacteria was quenched with trypan blue. Of the four strains tested via FACS analysis, two of which were mucoid and two nonmucoid, SP-D enhanced the uptake of only one nonmucoid strain. At 2 µg SP-D/ml, the uptake of strain V52385 was increased by 234 ± 75% compared with control. The uptake of the other nonmucoid strain (AS5093) was 127 ± 45%, and the uptake of mucoid strains J41036 and N66670 were 124 ± 33 and 127 ± 44%, respectively. Data shown are means ± SEM for three to five experiments.

To determine whether SP-D enhanced bacterial killing in the presence of macrophages, the CFUs obtained after incubation of bacteria for 60 min at 37°C in the presence or absence of SP-D were determined. SP-D had no detectable effect on the macrophage-mediated killing of the nonmucoid strain of P. aeruginosa. The number of CFUs recovered after incubation of the nonmucoid strain of P. aeruginosa with SP-D was 106 ± 24% (n = 6) of those recovered in the absence of SP-D. In contrast, SP-D decreased the number of CFUs recovered in the presence of SP-D when the mucoid strain of P. aeruginosa was present. In the presence of SP-D, the CFUs recovered were 85 ± 1.2% of those recovered in the absence of SP-D (n = 3).

Binding of SP-D to P. aeruginosa

As shown in Figure 4, SP-D bound to mucoid P. aeruginosa in a concentration- and calcium-dependent manner. Binding was inhibited by as little as 10 mM maltose (Figure 5). The magnitude of binding to the live mucoid P. aeruginosa was approximately 5-fold greater than the magnitude of binding to the heat-killed mucoid P. aeruginosa (data not shown). Both heat-killed and live nonmucoid bacteria bound SP-D in a calcium-dependent manner; the level of SP-D binding to the live nonmucoid bacteria was approximately 2-fold greater than to the heat-killed bacteria (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 4.   Binding of recombinant rat SP-D to live mucoid P. aeruginosa. Recombinant rat SP-D was radiolabeled with [3H]proline as described in MATERIALS AND METHODS. A total of 2 × 109 bacteria in 0.5 ml PBS containing 0.1% BSA and either 0.9 mM CaCl2 or 7 mM EDTA were incubated with various amounts of 3H-SP-D (0 to 2 µg) at room temperature with gentle shaking for 30 min, and then centrifuged at 6,000 × g for 5 min to precipitate the bacteria. Data shown are means ± SEM from three experiments. If error bars are not visible, they are smaller than the symbol.

View larger version (13K): [in this window] [in a new window]   Figure 5.   Binding of recombinant rat SP-D to live mucoid P. aeruginosa is inhibited by maltose. Binding was analyzed as described in Figure 4 legend.

Heat-Killing P. aeruginosa Affects Its Morphologic Appearance

Because the heat-killed and live P. aeruginosa bound SP-D to such different extents, the morphologic appearances of the bacteria were analyzed by electron microscopy. The appearance of the organism was dramatically altered by the heat treatment. Live bacteria (Figure 6A) possess a tightly layered cell envelope composed of an outer membrane and delicate cytoplasmic membrane. The cytoplasmic content is homogenous. A fine and relatively light network that is probably chromatin occupies the central portion of live cells. As shown in Figure 6B, the layered structure of the gram-negative envelope is preserved in heat-killed bacteria but the space between the outer and inner cytoplasmic membrane is often dilated. Dark cytoplasmic material is adherent to the inner surface of the inner membrane and thin, fibrillar chromatin material runs parallel to the axis of the cell.

View larger version (170K): [in this window] [in a new window]   View larger version (162K): [in this window] [in a new window]   Figure 6.   Electron micrographs of live (A) and heat-killed (B) mucoid P. aeruginosa. Samples were processed as described in Figure 1 legend. The morphologic appearance of the bacteria is dramatically affected by the heat-treatment. Original magnification: ×12,500.

SP-D Does Not Aggregate P. aeruginosa

One mechanism by which SP-D might enhance phagocytosis is by aggregation of the P. aeruginosa. However, when either mucoid or nonmucoid P. aeruginosa was incubated with 5 µg SP-D/ml for up to 2 h, no detectable aggregation (as assessed by a decrease in absorbance due to aggregate precipitation) was observed (Figures 7A and 7B). The bacteria also did not appear to be aggregated when examined by light microscopy (data not shown). In addition, lower concentrations of SP-D (0.25 and 1 µg/ml) also did not induce detectable bacterial aggregation (data not shown). The same preparation of SP-D was shown to aggregate E. coli (Figure 7C) in a calcium-dependent manner, consistent with previous observations by Kuan and colleagues (30).

View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 7.   Agglutination of bacteria by SP-D. Bacteria were diluted in PBS containing calcium and SP-D (5 µg/ml) or vehicle and the absorbance at 700 nm was measured periodically over 2 h. Aggregation is indicated by a decrease in absorbance as the bacteria settle out of solution. (A) Nonmucoid P. aeruginosa 28753; (B) mucoid P. aeruginosa S470; (C ) E. coli Y1088.

Mechanism of SP-D Enhanced by Phagocytosis

To evaluate the effects of SP-D as an opsonin, bacteria were preincubated with SP-D and then washed by centrifugation before addition to the macrophages. SP-D was a very effective opsonin; the percentage of macrophages containing bacteria in the absence of SP-D was 18 ± 3.5 compared with 35.3 ± 0.2% in the presence of SP-D (means ± SEM of three replicate determinations).

To test the possibility that SP-D may also stimulate phagocytosis by a direct interaction with the macrophage (e.g., by acting as an activation ligand), macrophages were adhered to slides precoated with lysine (as for the assays described earlier) or to slides that had been precoated with SP-D. As shown in Figure 8, adherence of macrophages to SP-D-coated slides did not significantly increase the phagocytosis of nonopsonized bacteria. These data suggest SP-D does not directly activate the macrophages to increase the uptake of P. aeruginosa. The uptake of P. aeruginosa preincubated with SP-D was not enhanced when the macrophages were adhered to SP-D-coated slides but was enhanced when the macrophages were adhered to lysine-coated slides. These data are consistent with the possibility that adherence of macrophages to SP-D-coated slides results in clustering of the SP-D receptors on the basolateral surfaces, making the receptors inaccessible to interact with SP-D-opsonized bacteria added to the apical surface.

View larger version (28K): [in this window] [in a new window]   Figure 8.   Effects of adherence of alveolar macrophages to lysine and SP-D-coated plates on SP-D-mediated phagocytosis. Isolated alveolar macrophages were allowed to adhere to lysine-coated or SP-D-coated LabTek chamber slides and then incubated with live mucoid P. aeruginosa or live mucoid P. aeruginosa that had been preincubated with SP-D, 5 µg/ml. After 60 min incubation, unbound bacteria were removed by washing and the fluorescence of extracellular bacteria was quenched with trypan blue. The percentage of macrophages with intracellular bacteria was quantitated by counting. Samples were coded so that the analysis was blinded.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results presented in this study demonstrate that SP-D acts as an opsonin to enhance the phagocytosis of the important pulmonary pathogen P. aeruginosa. Although SP-D bound to P. aeruginosa, it did not induce bacterial aggregation. Binding was calcium-dependent and inhibited by maltose. Macrophage-mediated killing of the mucoid strain of P. aeruginosa was enhanced slightly by SP-D.

The mechanism of enhancement of phagocytosis does not seem to be one of simple agglutination of P. aeruginosa inasmuch as we were unable to demonstrate either macroscopic or microscopic aggregation of P. aeruginosa by SP-D. These findings are in contrast to those reported by other investigators, who observed that SP-D induced aggregation of E. coli (30), A. fumigatus conidia (27), S. pneumoniae, and S. aureus (35).

Even though SP-D did not induce aggregation, it did bind to P. aeruginosa. The binding to P. aeruginosa was calcium-dependent and inhibited by saccharides, as was binding to E. coli (30) and A. fumigatus conidia (27). Thus, these studies suggest that binding of SP-D to an organism is not sufficient to induce aggregation.

SP-A and other collectins have been shown to act as activation ligands as well as opsonins (11, 45). Activation ligands are proteins that directly stimulate the cell to phagocytose; binding to the organisms (e.g., opsonization) is not required. To test the possibility that SP-D may act as an activation ligand, isolated alveolar macrophages were adhered to SP-D-coated chamber slides and then allowed to internalize either unopsonized P. aeruginosa or P. aeruginosa that had been preincubated with SP-D. Adherence to SP-D-coated slides did not enhance the uptake of unopsonized bacteria but did abrogate enhanced uptake seen with SP-D-opsonized bacteria. Thus, these results suggest that SP-D interaction with the macrophage alone is not sufficient to stimulate phagocytosis and are consistent with a role for SP-D as an opsonin. Hartshorn and coworkers (35) recently reported that SP-D enhanced uptake of several types of bacteria by neutrophils. They concluded that a major mechanism of SP-D-enhanced uptake by neutrophils was via aggregation of the bacteria with the SP-D. A direct interaction of SP-D and SP-A with the neutrophil was also reported.

Our data are also consistent with the possibility that adherence of macrophages to SP-D-coated slides clusters the SP-D receptors or binding sites on the basolateral surface of the cell, thereby making them inaccessible to interact with SP-D-coated organisms. The receptor(s) involved in modulating phagocytosis have not been identified, although previous studies have demonstrated that SP-D binds with high affinity to alveolar macrophages (23, 24), and Holmskov and coworkers reported the isolation of a glycoprotein of 340 kD (gp340) that was localized to alveolar macrophages and contains scavenger receptor domains (46).

Many phagocytosis studies have been carried out with heat-killed organisms to eliminate the complication of bacterial replication or the effects of bacterial metabolic products on cell activity. Because the effects of SP-A on phagocytosis of both heat-killed (14, 47) and live bacteria (17, 43) have been investigated, we thought it was important to make a direct comparison of the effects of SP-D on phagocytosis of both live and heat-killed P. aeruginosa. Our studies demonstrated that SP-D increased the uptake of both heat-killed and live P. aeruginosa. It has been previously demonstrated that heat-killed P. aeruginosa are phagocytosed more slowly than are live organisms by human polymorphonuclear leukocytes and that the post- phagocytic hexose monophosphate shunt activity varied with heat-killed versus live organisms (48). Although we did not carry out a detailed quantitative or time-dependent analysis, the magnitude of the stimulation by SP-D was fairly similar for both heat-killed and live organisms.

Although SP-A has been shown to enhance the phagocytosis of a variety of organisms, SP-A had little effect on phagocytosis of a heat-killed mucoid strain of P. aeruginosa by alveolar macrophages, consistent with our previous observations (43). However, we have recently found significant differences in the effects of SP-A on phagocytosis of heat-killed and live P. aeruginosa, and recent studies have shown that SP-A can stimulate the uptake of live P. aeruginosa in vitro (W. I. Mariencheck, unpublished observations). Studies by Manz-Keinke and coworkers (17) showed that the effects of SP-A on phagocytosis of P. aeruginosa vary with the growth phase of the organism. Together, these studies highlight the fact that subtle differences in the organism may have a profound effect on the phagocytic response. In this regard, it is important to note that SP-D enhanced the uptake of three of six strains tested. Thus, the effects of SP-D are dependent upon the strain and source of the organism.

Several reports have demonstrated that phagocytosis of P. aeruginosa is influenced by multiple experimental parameters, including the source of the macrophages, their state of maturation, and the concentration of glucose in the incubation buffer. For example, the uptake of P. aeruginosa by cultured human and mouse macrophages is increased by inclusion of 10 mM glucose in the incubation medium (49). In contrast, freshly isolated murine alveolar macrophages did not ingest P. aeruginosa even in the presence of glucose (49). Interestingly, glucose inhibits the binding of SP-D to alveolar macrophages (24). The concentration of glucose in the alveolar hypophase is estimated to be approximately one-fiftieth the concentration of glucose in the plasma (52). Although we did not make a systematic analysis of the role of glucose in SP-D-mediated phagocytosis, our data show that SP-D can enhance phagocytosis of P. aeruginosa by rat alveolar macrophages in the absence of added glucose and modestly increase bacterial killing in the presence of 2 mM glucose.

In vivo studies with SP-A-deficient mice and in vitro studies have confirmed a role for SP-A and alveolar macrophages in the response to P. aeruginosa infection. For example, in vitro SP-A enhances uptake of P. aeruginosa by alveolar macrophages (W. I. Mariencheck and J. R. Wright, unpublished observations), and macrophages from SP-A- deficient mice ingested significantly fewer organisms than did macrophages from wild-type mice at 1 h after infection (53). In addition, an earlier influx of polymorphonuclear leukocytes occurred in the SP-A-deficient lungs infected with P. aeruginosa. Although the role of the alveolar macrophage in controlling the infection was not elucidated in this study, Kooguchi and colleagues (54) reported that depletion of macrophages has a beneficial effect on measures of early lung injury but a deleterious late effect on lung injury and survival in a wild-type mouse model of P. aeruginosa infection. The roles of SP-D and macrophages in regulating P. aeruginosa infection in vivo await investigations with the recently described SP-D-deficient mouse (21, 22), although this model is complicated by the fact that SP-A levels can be elevated in the presence of an SP-D deficiency (21).

SP-D Levels in Disease States

Patients with CF are susceptible to infections with P. aeruginosa. The factors that predispose to this susceptibility are complicated and not well understood. It is tempting to speculate that decreases in SP-D levels in CF may facilitate the infectivity of P. aeruginosa. In fact, it has been reported that SP-A levels were decreased in bronchoalveolar lavages of patients with CF (55) and it was recently reported in abstract form that levels of SP-D were decreased as well (56). Although these observations do not distinguish cause from effect, it is intriguing to speculate that a deficiency in SP-D may contribute to the chronic infection often seen in patients with CF and that, more generally, changes in the surfactant system may be important in a variety of disease states.