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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 249-256, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4896

Surfactant Protein A and D Differently Regulate the Immune Response to Nonmucoid Pseudomonas aeruginosa and Its Lipopolysaccharide

Philip Bufler, Bettina Schmidt, Daniela Schikor, Adolf Bauernfeind, Erika C. Crouch and Matthias Griese

Dr. von Haunersches Kinderspital, University of Munich, Munich, Germany; Department of Pathology, Washington University, School of Medicine, St. Louis, Missouri; and Max von Pettenkofer Institut, University of Munich, Munich, Germany

Address correspondence to: Matthias Griese, Dr. von Haunersches Kinderspital, University of Munich, Pettenkoferstrasse 8a, D-80336 Munich, Germany. E-mail: griese{at}pk-i.med.uni-muenchen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of the surfactant proteins (SPs) A and D in the pulmonary immune defense of nonmucoid strains of Pseudomonas aeruginosa, the most etiologic agents of nosocomial Pseudomonas pneumonia. We first examined the interactions of recombinant human SP-D dodecamers and purified natural or recombinant human SP-A with two smooth, and two rough, clinical isolates of nonmucoid P. aeruginosa. SP-D bound to all four isolates, but agglutinated only one rough and one smooth strain. SP-D functioned as an opsonin to enhance the uptake of all four strains by the human monocytic cell line Mono Mac 6 (MM6). SP-D also enhanced tumor necrosis factor-{alpha} secretion by MM6 cells in response to purified lipopolysaccharide (LPS) isolated from the rough, but not the smooth, strains. Although SP-A bound to all four strains, it did not cause bacterial aggregation or enhance uptake. It showed small but statistically significant inhibitory effects on the cytokine response of MM6 cells to one strain of smooth organisms, but did not significantly alter the response to purified LPS. This study in combination with previously published data strongly suggests that SP-D may play important roles in the local innate pulmonary defense against nonmucoid P. aeruginosa of diverse LPS phenotypes, and preferentially augments the cellular response to rough P. aeruginosa endotoxin.

Abbreviations: colony-forming units, cfu • ethylenediamine tetraacetic acid, EDTA • fetal calf serum, FCS • fluorescein isothiocyanate, FITC • lipopolysaccharide, LPS • Mono Mac 6, MM6 • phosphate-buffered saline, PBS • surfactant protein, SP • Tris-buffered saline, TBS • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hydrophilic surfactant proteins (SPs) A and D are oligomeric glycoproteins that belong to the family of calcium-dependent, collagenous C-type lectins ("collectins"). These collectins appear to be involved in innate, first-line immunity (for review see Refs. 1, 2). SP-A and SP-D are synthesized in the lungs by alveolar type II cells and Clara cells, and are secreted into the alveoli and distal airways (3, 4).

Both SP-A and SP-D can interact with a variety of pulmonary pathogens. Specific binding of these collectins has previously been demonstrated to gram-negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae) or gram-positive bacteria (Streptococcus pneumoniae, Staphylococcus aureus), Mycobacterium tuberculosis, viruses (influenza A and respiratory syncytial virus), fungi (Cryptococcus neoformans, Aspergillus fumigatus), and Pneumocystis carinii. The interaction of SP-A and SP-D with the microbe usually occurs via the lectin domain, and C-type lectin dependent binding strongly depends on the presence of Ca2+. Binding of SP-A and SP-D can be associated with agglutination of the pathogen; however, SP-D is the more potent agglutinating agent (1).

Previous studies have described various receptors for SP-A and SP-D on alveolar macrophages (5–7). Although their relevance for signal transduction has not been fully elucidated, SP-A and SP-D modulate the host defense functions of alveolar macrophages and circulating leukocytes. For example, SP-A and SP-D can stimulate the phagocytosis of bacteria and viruses, the production of oxygen radicals, and leukocyte chemotaxis (2). There are conflicting data concerning the ability of SP-A to modulate the stimulation of leukocytes with bacteria or lipopolysaccharide (LPS). Several authors reported that SP-A reduced the cytokine release of smooth LPS-stimulated human alveolar macrophages (8), human buffy coat cells (9), or the human macrophage cell line U937 (10). Consistent with these data, in vivo experiments showed that SP-A–deficient mice intratracheally challenged with smooth Escherichia coli 026:B6 LPS produced significantly more tumor necrosis factor (TNF)-{alpha} than the wild-type mice (11). Contrary results were published by Phelps and coworkers, who demonstrated that purified proteinosis SP-A can stimulate TNF-{alpha} secretion by THP-1 cells (12).

P. aeruginosa is an important cause of noscomial pneumonia and sepsis, and an important pulmonary pathogen in cystic fibrosis. Nonmucoid strains of this organism are responsible for most cases of nosocomial Pseudomonas pneumonia. Ineffective clearance of nonmucoid bacteria can result in the proliferation of bacteria with a mucoid, alginate-producing phenotype. In addition, chronic endobronchial infection by P. aeruginosa can be associated with changes in the O-antigen composition of its LPS. In particular, the LPS synthesized by the bacteria can convert from the smooth to a rough serotype expressing few short O-linked side chains (13). Levels of SP-A and SP-D have been shown to be decreased in the lungs of patients with cystic fibrosis (14–16).

Recent studies suggest that SP-A and SP-D are involved in the defense against P. aeruginosa. SP-A was shown to increase the uptake of a live, mucoid strain of P. aeruginosa (17). Furthermore, the clearance of an intratracheally administered mucoid strain of P. aeruginosa was significantly reduced in SP-A–deficient mice (18). In another study, SP-D was shown to stimulate the phagocytosis of some mucoid and one nonmucoid strains of P. aeruginosa by alveolar macrophages (19). However, the LPS phenotype and the effects of collectins on the macrophage cytokine response to these organisms or their LPS have not been specifically examined in vitro or in vivo.

The interactions of the collectins with LPS could be influenced by differences in the binding mechanism. For example, SP-A has been shown to bind to the lipid A domain of E. coli LPS (20), and preferentially interacts with organisms bearing rough LPS with truncated O-antigens. By contrast, previous studies using E. coli and Salmonella minnesota LPS suggest that SP-D preferentially interacts with the core oligosaccharide of rough forms of LPS (21). However, different gram-negative organisms show variations in the extent and type of O-polysaccharide substitution, as well as more subtle differences in the structure of the conserved core oligosaccharide domains. These differences could influence interactions with various binding proteins and host cells.

The aim of this study was to examine the potential roles of SP-A and SP-D in the pulmonary immune defense against nonmucoid strains of P. aeruginosa, and to examine the potential influence of the Pseudomonas LPS phenotype on the inflammatory response of mononuclear phagocytes. We used the human monocytic cell line MM6, which express phenotypic and functional features of mature monocytes (22), as a convenient and reproducible model for human alveolar macrophages, and we analyzed four clinical isolates of nonmucoid P. aeruginosa with distinct LPS serotypes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
All reagents were obtained from Sigma-Aldrich Vertriebs GmbH (Deisenhofen, Germany) unless indicated otherwise. For LPS measurements the Limulus-Amoebocyte-Lysat (LAL)-kinetic-QCL assay kit (Bio-Whittaker, Walkersville, MD) was used.

Bacteria, Cells and Cell Culture
Bacteria. Clinical isolates of P. aeruginosa from stable, but chronically infected, patients with cystic fibrosis were serotyped using O-antigen–specific antisera, grown on tryptone soya agar plates (Oxoid, Basingstoke, Hampshire, UK) and harvested after overnight growth at 37°C. The pyocin type was routinely obtained as an epidemiologic marker of P. aeruginosa.

Fluorescein isothiocyanate labeling of bacteria. Overnight cultures of bacteria on agar plates were suspended in phosphate-buffered saline (PBS, pH 7.2). After centrifugation, the bacteria were resuspended at an OD623 nm of 1.5 (1.5 x 109 colony forming units [cfu]/ml) and sedimented again. Then the bacteria were resuspended in 0.1 M sodium-carbonate buffer, pH 9.0. Fluorescein isothiocyanate (FITC; Sigma-Aldrich), prepared in low-endotoxin dimethyl sulfoxide at 10 mg/ml, was added to a final concentration of 0.1 mg/ml. The bacteria were incubated on a rotation cycler for 1 h at room temperature in the dark. FITC-stained P. aeruginosa were washed four times in PBS, and aliquots were stored at -80°C (23).

Cell culture. The human monocytic cell line Mono Mac 6 (MM6), kindly provided by H.-W. L. Ziegler-Heitbrock (Institute for Immunology, University of Munich, Germany), was grown in RPMI 1640 (Biochrom KG, Berlin, Germany), ultrafiltered through a Gambro 2,000 column (Gambro, Hechingen, Germany) to eliminate LPS contamination. RPMI was supplemented with 10% heat inactivated fetal calf serum (FCS; Biochrom KG), 100 U/ml penicillin, 0.1 mg/ml of streptomycin, 1% amino acids (all Biochrom) and OPI-supplement (Sigma-Aldrich). Differentiation of MM6 into the macrophage lineage was induced by pretreatment with Vitamin D3 (Biomol, Plymouth Meeting, PA) (107 M, 48 h) or LPS (Salmonella minnesota, 10 ng/ml, 48 h) as described by others (24). Differentiation of MM6 cells with LPS gave best results in phagocytosis of bacteria, and pretreatment with Vitamin D3 was optimal to increase the response of MM6 cells to LPS and stimulation by P. aeruginosa. The mouse fibroblast cell line A9, kindly provided by H. Engelmann (Institute for Immunology, University of Munich), was grown in RPMI supplemented with 5% heat inactivated FCS (Biochrom KG), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1 mM sodiumpyruvate, 2 mM N-acetyl-L-alanyl-L-glutamine (all Biochrom KG), and was used to measure TNF-{alpha} activity in the supernatant of stimulated MM6 cells. All cells were cultured in a water saturated air-atmosphere with 6% CO2 at 37°C.

Surfactant Proteins A and D
Recombinant human SP-D in Tris-buffered saline (TBS) containing 10 mM ethylenediamine tetraacetic acid (EDTA) was produced in Chinese hamster ovary cells (25). The protein was isolated from the culture medium by maltosyl-agarose chromatography, and dodecamers were purified by gel filtration chromatography under nondenaturing conditions (25). The purity of the protein was confirmed by SDS-PAGE and silver staining. It was active as a viral agglutinin and enhanced virus uptake by neutrophils. SP-A, isolated from the bronchoalveolar lavage of patients with alveolar proteinosis, was a gift of H. P. Haagsman (Department of Biochemistry, Cell Biology, and Histology and Graduate School of Animal Health, University Medical Center Utrecht, The Netherlands) (26), and used for phagocytosis studies. Recombinant human SP-A, produced in Chinese hamster ovary cells and used for cytokine stimulation assays, was kindly provided by W. Steinhilber, Byk Gulden, Konstanz, Germany. Endotoxin monitoring was negative (< 0.005 ng/ml) for all SP-A preparations. Minor endotoxin contamination of 50 pg LPS/µg SP-D was detected for SP-D. Thus, 500 pg LPS was introduced into the assay system together with the SP-D. This amount of purified P. aeruginosa–LPS used in this study had no effect on TNF-{alpha} production, when tested alone.

Isolation of P. aeruginosa LPS
LPS from P. aeruginosa was extracted by the phenol method according to Westphal and coworkers (27). Freshly grown bacteria were stirred in 90% aqueous phenol at 68°C for 1 h. After cooling on ice, the suspension was centrifuged (1,700 x g, 60 min). The aqueous phase containing LPS was separated and extensively dialyzed against H2O to remove phenol. Contaminating nucleic acids were precipitated by the addition of 6% cyteltriomethylammoniumbromide (Serva Electrophoresis GmbH, Heidelberg, Germany) at 4°C overnight. Precipitated nucleic acids were removed by centrifugation (1,700 x g, 60 min). NaCl was added to the supernatant to a final concentration of 1 M. Then the LPS was precipitated two times by the addition of ten volumes of ethanol 96% at 4°C for 3 d. After centrifugation (12,000 x g, 30 min) the precipitated LPS was dissolved in 1 M NaCl, dialyzed against H2O, and dried in a vacuum dessicator. Purified LPS was dissolved in H2O and vigorously vortexed before use. All LPS preparations were analyzed for purity and maturation of the O-linked polysaccharide side chains by gel electrophoresis (Figure 1) .



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  		Figure 1. Silver-stained 10% NuPAGE Bis-Tris gel of LPS purified form the indicated clinical isolate of P. aeruginosa. Samples of 1 µg LPS per lane were applied. According to the serotype, the proportion of O-antigen is highest for P. aeruginosa 5 and 11.

 
Bacterial Agglutination Assay
For agglutination equal volumes of bacterial suspensions in TBS with 2 mM calcium (TBS/Ca) (50 µl, OD623nm = 0.1) were incubated with SP-A and SP-D (10 µg/ml) at room temperature and immediately examined on a glass slide for macroscopic or microscopic agglutination. Aliquots of the reaction mixture were dried on glass coverslips and gram-stained for photographic documentation. Control experiments were performed in the presence of maltose or glucose (100 mM), or EDTA (10 mM).

Binding of SP-A and SP-D to P. aeruginosa
This binding assay was performed essentially as published by Hartshorn and colleagues, who studied SP-D binding to E. coli or S. pneumoniae (28). Microtiter plate wells (Maxisorp; Nunc, Kamstrup, Denmark) were coated with suspensions of live P. aeruginosa (OD623nm = 0.1, 100 µl/well) in carbonate buffer (Na2HCO3 15 mM, NaHCO3 35 mM, pH 9.6; Merck Eurolab GmbH, Ismaning, Germany) and allowed to dry at 37°C. Nonspecific binding sites were blocked with 1% gelatin in TBS/Ca for 4 h at room temperature (150 µl/well). After washing with TBS/Ca adherent bacteria were incubated with serial dilutions (0–10 µg/ml) of SP-A and SP-D in TBS/Ca with or without the addition of EDTA 10 mM. After 1 h incubation at room temperature, the plates were washed three times with TBS/Ca and glutaraldehyde 0.25% in TBS/Ca was added for 15 min. Reactive sites were blocked with bovine serum albumin 1% in TBS/Ca. Polyclonal antibodies against SP-A (gift of W. Steinhilber, BykGulden) and SP-D (1 µg/ml in TBS/Ca/bovine serum albumin 1%) (gift of K.B. Reid, Department of Biochemistry, University of Oxford, UK [29]) were then added to each well (100 µl/well) and incubated for 2 h at room temperature. The plates were washed three times in TBS/Ca and alkaline-phosphatase–conjugated goat anti-rabbit IgG (Dianova GmbH, Hamburg, Germany) was added for 1 h at room temperature. After three washes with TBS/Ca, 100 µl/well of ABTS reagent (Boehringer Mannheim GmbH, Mannheim, Germany) was added and incubated until sufficient color developed. The plates were read at 405 nm. A negative signal was obtained for the wells incubated with SP-A and SP-D (10 µg/ml) in the absence P. aeruginosa. Nonspecific binding was defined as binding in the presence of 10 mM EDTA. Specific binding was calculated by subtraction of nonspecific binding from total binding. Maximum binding (plateau of the binding curve) was observed at 1,000 ng/ml of SP-A and 100 ng/ml of SP-D. In Table 1, binding is expressed in a semiquantitative manner assessing the relative binding of SP-A or SP-D to the different strains of P. aeruginosa at maximal binding. Due to possible differences in the extent or affinity of antibody binding it is not possible to accurately compare the binding signal obtained for the two collectins.


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  		TABLE 1 Characterization of P. aeruginosa

 
Phagocytosis Assay
Freshly thawed FITC-stained bacteria in PBS (1 x 107 cfu/assay) were opsonized with surfactant protein or normal human serum (pooled of five healthy donors) for 60 min as indicated. LPS-treated MM6 cells in RPMI (0.5 x 106 cells/sample) were added to a final volume of 50 µl. Free CaCl2 concentration was adjusted to 2 mM. The concentration of glucose in RPMI did not significantly affect the phagocytosis. The final concentration of surfactant protein and human serum was 10 µg/ml and 2%, respectively. After the incubation for 1 h at 37° the phagocytosis was terminated with 1 ml of cold PBS/Ca 2 mM. After washing three times in cold PBS/Ca, the pellet was resuspended in 50 µl PBS/Ca. The extracellular fluorescence was quenched with the addition of 50 µl trypan blue solution (1 mg/ml wt/vol in PBS/Ca) for 2 min on ice. After washing, the cells were immediately analyzed by fluorescence-activated cell sorting. For each assay cells were incubated with fluorescent bacteria at 4°C to demonstrate adequate quenching as controlled by fluorescence microscopy.

Stimulation of MonoMac6 Cells with LPS or P. aeruginosa
P. aeruginosa–LPS (10 µg/ml final concentration per assay) or vital P. aeruginosa (1 x 107 cfu/ml) from overnight growth on agar plates were preincubated with SP-A or SP-D at different concentrations in MM6-culture medium containing 10% FCS for 15–30 min at 37°C. For the SP-D, which contained 10 mM EDTA, the medium was additionally supplemented with CaCl2 to a final concentration of 2 mM of free calcium. Ionized calcium and the pH of the medium conditioned in an atmosphere of 6% CO2 was proved to be in the physiologic range by a blood gas analyzer. Vitamin D3–treated MM6 cells were then added to a final concentration of 0.5 x 106 cells/ml in a volume of 120 µl in 96-well microtiter plates, and incubated at 37°C for 6 h. After centrifugation for 10 min, the supernatant was collected and stored at –80°C for cytokine measurement.

TNF-{alpha} Assay
The TNF-{alpha} activity in the supernatant of stimulated MM6 cells was determined using a bioassay as described (30). Briefly, the murine fibroblast cell A9 was seeded in 96-well plates 18 h before assay, at a density of 25,000 cells/well in 100 µl medium. Serial dilutions of TNF-{alpha} standard (50–0.01 U/ml), kindly provided by Dr. H. Engelmann (Institute for Immunology, University of Munich), and samples were then applied together with 50 µg/ml cycloheximide. After 18 h, the cells were washed and viability was assessed by measuring neutral red uptake.

Statistical Analysis
Statistical Analysis was performed with Prism 3.0 (Graph Pad Software, San Diego, CA). Differences among experimental groups were determined by performing ANOVA. The modified t test according to Newman-Keuls was applied as a multiple comparison procedure. A P value of < 0.05 was considered significant. Values are expressed as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SP-D–Mediated Agglutination of P. aeruginosa Is Not Predicted by the LPS Serotype
We characterized four clinical isolates of P. aeruginosa with respect to the LPS-serotypes, maturation of LPS, pyocin-types, and mucin production (Table 1). To test whether SP-A and SP-D interact with these strains of P. aeruginosa, we initially performed agglutination assays. Two strains of P. aeruginosa were agglutinated by SP-D (Table 1, Figure 2) . However, no agglutination was seen in the presence of SP-A up to a maximum concentration of 20 µg/ml. The carbohydrate specificity of SP-D–mediated agglutination was assessed by repeating the experiments in the presence of EDTA and selected carbohydrate ligands of SP-D (data are only shown for Ps. 12, Figure 2). The SP-D–mediated agglutination of P. aeruginosa required free divalent cations and was inhibited by maltose, glucose, and mannose. Interestingly, SP-D–mediated agglutination was observed both for rough and smooth P. aeruginosa (Table 1). The minimal SP-D concentration required for agglutination was 2 µg/ml. The absence of agglutination was not attributable to the absence of binding because all strains of P. aeruginosa specifically bound to SP-A and SP-D (Table 1).



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  		Figure 2. Light microscopy of Ps. 12 (OD623nm = 0.1) after treatment with SP-D (10 µg/ml) in TBS/Ca++ 2 mM for 5 min (A). Agglutination was reversed by the addition of EDTA 10 mM (B), maltose (100 mM) (C), or glucose (100 mM) (D). Aliquots of the reaction mixture were dried on glass coverslips and Gram-stained for photo documentation. Original magnification: x400.

 
SP-D but Not SP-A Stimulates Phagocytosis of Nonmucoid P. aeruginosa by MM6 Cells
To determine whether differences in agglutinatibility of P. aeruginosa by SP-A and SP-D can alter the opsonic activity, we performed phagocytosis assays with the four nonmucoid P. aeruginosa strains used for agglutination studies. To ensure opsonization, the bacteria were pretreated with SP-A, SP-D, or human serum 30 min before the assay was started. Opsonization with human serum significantly increased phagocytosis of all four strains of P. aeruginosa by MM6 cells (Figure 3) . Opsonization with SP-D similarly stimulated phagocytosis of the agglutinating and nonagglutinating strains of P. aeruginosa by 2- to 3-fold at a concentration of 10 µg/ml in the absence of serum (Figure 3). Interestingly, SP-A did not alter the phagocytic uptake at concentrations up to 20 µg/ml. The combination of SP-D or SP-A with human serum also did not result in an additional increase of phagocytosis. Phagocytosis required the presence of Ca2+. Although some effects of SP-A on phagocytosis are nonopsonic and involve direct interactions with the phagocyte (31), preincubation of MM6 cells with SP-A or SP-D had no effect on bacterial uptake (data not shown). Taken together, collectin effects on uptake were only observed after opsonization of the bacteria; there was no direct enhancement of macrophage phagocytosis by SP-A or SP-D.



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  		Figure 3. SP-D but not SP-A stimulates phagocytosis of P. aeruginosa by Mono Mac 6 cells. LPS-treated MM6 cells were incubated with four different clinical isolates of live, nonmucoid FITC-stained P. aeruginosa, e.g., P. aeruginosa 5 (dotted bars), 11 (open bars), 12 (striped bars), and 20 (solid bars), for 1 h at a cell/bacteria ratio of 1:20. Bacteria were pretreated with human serum (2%), or with SP-A or SP-D (10 µg/ml). The extracellular fluorescence was quenched with the addition of trypan blue. After washing, the cells were immediately analyzed by fluorescence-activated cell sorter. The results are expressed as the percentage of cells with higher fluorescence than untreated cells. Each column represents the mean of three to five independent experiments, and statistical analysis was performed by ANOVA and Newman-Keuls post hoc test using the pooled data of all strains of P. aeruginosa. ***P < 0.001 compared with control.

 
SP-D and SP-A Differently Regulate Induction of TNF-{alpha} Production in Response to Nonmucoid P. aeruginosa
Binding of SP-A and SP-D to P. aeruginosa might also modulate the cytokine response of macrophages to these bacteria. To test this hypothesis MM6 cells were incubated with vital P. aeruginosa with or without SP-A and SP-D at a maximum concentration of 10 µg/ml for 6 h. The TNF-{alpha} activity in the culture supernatant was then analyzed. All four strains of P. aeruginosa stimulated TNF-{alpha} secretion by MM6 cells in the absence of SP-A or SP-D (Table 2). SP-A slightly inhibited the TNF-{alpha} secretion by MM6 cells in one strain of P. aeruginosa with a smooth LPS serotype by 20–25% (Figure 4 , top panel). In contrast, SP-D significantly increased the TNF-{alpha} secretion induced by Ps. 11 up to 150% of control and slightly increased the TNF-{alpha} release induced by strains 5 and 20, but significantly reduced Ps. 12–induced TNF-{alpha} secretion (Figure 4, bottom panel). Thus, the effects of SP-D are modest and did not correlate with bacterial agglutination.


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  		TABLE 2 Stimulation of TNF-{alpha} secretion by P. aeruginosa and its LPS

 


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  		Figure 4. SP-D and SP-A differently regulate P. aeruginosa–induced TNF-{alpha} production. Differentiated MM6 cells (0.5 x 106/ml) were stimulated for 6 h with vital P. aeruginosa (1 x 107 cfu/ml) in the presence or absence of SP-A or SP-D as indicated (open bars, 1 µg/ml; lightly shaded bars, 5 µg/ml; darkly shaded bars, 10 µg/ml). For SP-D containing 10 mM of EDTA the medium was additionally supplemented with CaCl2 to a final concentration of 2 mM. After incubation for 6 h at 37°C, TNF-{alpha} secretion into media was measured by A9 cell bioassay and expressed as percent of control. Results are means of three independent experiments and a P value of <0.05 of the ANOVA was considered significant. The results of the post hoc comparisons between the controls and those with SP-A or SP-D are indicated above the columns. *P < 0.05, **P < 0.01, ***P < 0.001.

 
Modulation of P. aeruginosa–LPS-Induced TNF-{alpha} Release by SP-D Is Dependent on the LPS Serotype
LPS is a major antigenic component of the outer surface of P. aeruginosa and contributes to the activation of macrophages. Therefore, we next investigated whether purified LPS can mimic the effect of vital P. aeruginosa upon TNF-{alpha} secretion from MM6 cells. MM6 cells were stimulated for 6 h with purified LPS isolated from P. aeruginosa (Figure 1) in the presence or absence of SP-A or SP-D. As observed for the bacteria, all LPS preparations markedly stimulated TNF-{alpha} secretion by MM6 cells (Table 2). Markedly less TNF-{alpha} stimulation was observed for the preparations of rough LPS isolated from Ps. 12 and Ps. 20. SP-A treatment did not significantly alter the TNF-{alpha} secretion induced by smooth or rough LPS (Figure 5) . This was in contrast to SP-D, which significantly enhanced the TNF-{alpha} release secondary to rough LPS isolated from Ps. 12 and Ps. 20. Although SP-D slightly but significantly inhibited the TNF-{alpha} release induced by smooth LPS isolated from Ps. 5 (serotype O11), it did not modulate cytokine secretion in response to Ps. 11-LPS (serotype O10) (Figure 5).



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  		Figure 5. SP-D action upon TNF-{alpha} release of MM6 cells induced by P. aeruginosa–LPS depends on the O serotype. Differentiated MM6 cells (0.5 x 106/ml) were incubated for 6 h with P. aeruginosa–LPS (10 µg/ml) in the presence or absence of SP-A or SP-D at different concentrations as indicated (open bars, 1 µg/ml; lightly shaded bars, 5 µg/ml; darkly shaded bars, 10 µg/ml). TNF-{alpha} secretion into media was measured by A9 cell bioassay. LPS was purified from the indicated strain of P. aeruginosa as described in MATERIAL AND METHODS. Means of three independent experiments are shown and a P value of <0.05 in the ANOVA was considered significant. The results of the post hoc comparisons between the controls and those with SP-A or SP-D are indicated above the columns. ***P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that SP-A and SP-D have different effects on the macrophage response to nonmucoid P. aeruginosa and its LPS. We demonstrated that SP-D, but not SP-A, stimulates the uptake of nonmucoid P. aeruginosa by the human monocytic cell line MM6. This uptake requires prior opsonization, but does not involve direct cellular activation, and does not require bacterial agglutination. The lung collectins showed relatively small effects on the cytokine response to living P. aeruginosa. The TNF-{alpha} response to one strain of vital P. aeruginosa was slightly inhibited by SP-A, and SP-D stimulated the cellular response to both of the smooth strains and one of the rough strains by < 2-fold. Although SP-A did not alter TNF-{alpha} response in response to purified rough or smooth P. aeruginosa LPS, the TNF-{alpha} response to rough LPS was selectively and quite markedly increased in the presence of SP-D.

Previous studies have shown that SP-A and SP-D, which have multiple trimeric lectin domains, are able to mediate bridging interactions between various microorganisms, including bacteria, fungi, and viruses (19, 21, 23, 32). Agglutination could facilitate the mechanical removal of bacteria from the lungs by the mucociliary clearance and could also increase the phagocytosis of bacteria. In the case of influenza virus, agglutination enhances the internalization by neutrophils via cellular receptors recognized by the virus (33). As others, we could not detect agglutination of P. aeruginosa by SP-A (17, 23). However, this is the first study to demonstrate agglutination of vital P. aeruginosa cells by SP-D, and the first to demonstrate efficient lectin-dependent agglutination of a smooth gram-negative bacterium. The agglutination of P. aeruginosa by SP-D depended on the presence of Ca++ or divalent cations, and was inhibited by competing saccharide ligands, consistent with involvement of the SP-D carbohydrate recognition domain. However, agglutination of P. aeruginosa by SP-D was not reliably predicted by the pattern of maturation of the LPS expressed on the bacterial surface.

Although only two of the strains showed collectin-mediated agglutination, SP-A and SP-D specifically bound to all P. aeruginosa strains used in this study via their lectin-binding domain in a concentration-dependent manner (Table 1). At present, LPS is the only known ligand for SP-A and SP-D on gram-negative organisms, and LPS inhibits SP-D–dependent agglutination of several gram-negative bacteria, including E. coli and Klebsiella pneumoniae (21, 34). Binding of the SP-D lectin domain to P. aeruginosa has also been demonstrated (35). But additional factors, e.g., the presence of other cell wall glycoconjugates, structural differences in the LPS, or the density and microorganization of the LPS are likely to play a role to induce bacterial agglutination by SP-A or SP-D. Although the binding of SP-A and SP-D to P. aeruginosa did not differ between P. aeruginosa expressing smooth or rough LPS, agglutination was associated with an enhanced binding signal for SP-D (Table 1). This could be of relevance in the clinical setting; P. aeruginosa can change the expression of O-linked oligosaccharides of LPS in the context of cystic fibrosis (36). Our findings suggest that this alteration of the LPS phenotype may not necessarily alter the interactions with SP-A or SP-D.

SP-A and SP-D have been shown to enhance the phagocytosis of bacteria as well as viruses and some fungi (for review see Ref. 1). Opsonization of the bacteria with SP-D increased the phagocytosis of all strains of P. aeruginosa used in this study by MM6 cells to a similar extent as opsonization with human serum. Recently, Restrepo and coworkers reported that SP-D could enhance the phagocytosis of 1 of 3 mucoid and 2 of 3 nonmucoid strains of vital P. aeruginosa by rat alveolar macrophages; these effects were not accompanied by bacterial agglutination and appeared to involve a specific macrophage receptor (19). Together with our findings, this suggests that other ligands than LPS or the spatial organization of LPS on the surface of P. aeruginosa may contribute to SP-D induced phagocytosis. Alternatively, there may be structural differences in the LPS expressed by the various strains of P. aeruginosa. For example, recent studies have shown that SP-D can bind to the O-antigens of specific serotypes of unencapsulated Klebsiella pneumoniae that contain repeating units of mannose, an SP-D ligand (H. Sahly, I. Ofek, and E. Crouch, unpublished data).

In contrast to previous results published by others using different cell systems as alveolar macrophages (17, 37) or THP-1 cells (38), SP-A did not stimulate the phagocytosis of vital P. aeruginosa. Similarly, in SP-A knockout mice decreased phagocytosis and pulmonary clearance of a mucoid strain of P. aeruginosa was reported (39). Notably, none of the P. aeruginosa strains used for these studies was characterized according to the LPS serotype and additional strain variations, including the effects of alginate production, or differences in the phenotype of the phagocytic cells cannot be excluded. Because Manz-Keinke and coworkers demonstrated that SP-A similarly enhances the phagocytosis of P. aeruginosa growing logarithmically or harvested in the stationary phase, it is unlikely that our finding can be explained by differences in the growth phase of P. aeruginosa (37). It is also possible that differences in the specific SP-A preparation played a role; however, the SP-A used for these studies was functional in other in vitro studies with respect to phagocytosis (31, 37), and bound in a lectin-dependent manner to all four strains of nonmucoid organisms used in our study.

Inflammation at the site of infection involves the secretion of proinflammatory cytokines. The production of TNF-{alpha} by activated macrophages is beneficial to eliminate infectious agents, but overproduction could worsen the disease state. The interaction of SP-A and SP-D with P. aeruginosa in the lung may alter this immune response. Our studies have shown that SP-A and SP-D can modulate TNF-{alpha} release by MM6 cells in response to vital nonmucoid P. aeruginosa, and demonstrated that SP-A and SP-D differently regulate the P. aeruginosa–induced TNF-{alpha} release. Although there were no major effects of SP-A on the response of these cells to vital organisms, SP-A slightly inhibited the TNF-{alpha} secretion by MM6 cells in one strain of P. aeruginosa at a concentration of 10 µg/ml. This is consistent with the study of Levine and coworkers, who demonstrated increased levels of cytokines, including TNF-{alpha}, upon challenge of SP-A knockout mice with mucoid P. aeruginosa (18). An inhibitory effect of SP-A upon vital P. aeruginosa–mediated TNF-{alpha} secretion was also demonstrated by Hickling and colleagues using buffy coat cells (9).

Given the previously published data, we were surprised to observe such modest effects of SP-A on the cytokine response to smooth and rough P. aeruginosa. Because all bacteria bound SP-A to about the same extent, it is possible that SP-A binding to P. aeruginosa is necessary, but not a sufficient prerequisite for inhibition. Additional signals differing between various strains of P. aeruginosa, including the specific LPS phenotype, or even differences in the target cells may be of importance. For example, Sano and coworkers demonstrated that SP-A does not bind to smooth LPS (E. coli 026:B6), but markedly inhibits the smooth LPS-induced TNF-{alpha} secretion by U937 (10). Because SP-A can bind to the cellular coreceptor for LPS (mCD14), they proposed that this interaction may prevent smooth LPS binding to mCD14.

In contrast to SP-A, SP-D stimulated the TNF-{alpha} release induced by three of the four strains of P. aeruginosa. As demonstrated for SP-A, this effect did not correlate with the extent of SP-D binding to P. aeruginosa or agglutination.

LPS is the major immunoreactive and biologically active component of the outer membrane of P. aeruginosa, and we were interested in whether LPS may completely mimic the effect of vital P. aeruginosa. We therefore performed the cytokine assays with both the living P. aeruginosa and their isolated LPS. However, there was no direct relationship in the response to organisms or the corresponding purified LPS. SP-A showed little effect on the response to bacteria or purified LPS; there was only a slight inhibition for two of the strains. In contrast, SP-D significantly enhanced TNF-{alpha} secretion in response to rough P. aeruginosa–LPS, but did not increase the cytokine response to smooth LPS. This effect of SP-D upon LPS-induced TNF-{alpha} release did not correlate with the response obtained with the corresponding live bacteria. These findings suggest that the effects of SP-D on the cytokine response to purified P. aeruginosa LPS are dependent on a rough LPS phenotype, but that the effects on intact organisms are more complex. The effects of SP-D on cytokine expression could reflect differences in the presentation of LPS to the target cells or to LPS-binding proteins present in the serum. Alternatively, the effects might be mediated by direct interactions with other molecules involved in LPS signal transduction such as CD14 (10). This interaction might change the association of bacterial products like LPS to immune cells and thus modulate the release of proinflammatory cytokines.

The concentrations for SP-A and SP-D in the epithelial lining fluid in vivo are presently unknown and still under investigation. SP-A and SP-D concentrations in the bronchoalveolar lavage fluid of healthy individuals were reported to be 4.6–12.4 µg/ml and 641–880 ng/ml for SP-A and SP-D, respectively (2, 14, 15, 16, 40). The components of the alveolar lining fluid are assumed to be diluted several times in the fluid recovered after bronchoalveolar lavage. Accordingly, the surfactant protein concentrations used in this study are likely to be in the physiologic range. Similar concentrations of SP-A and SP-D were used in previous reports.

In conclusion, it is likely that SP-A and SP-D play distinct roles in the local pulmonary defense of P. aeruginosa. Although previous in vitro studies have shown that SP-D preferentially interacts with the core oligosaccharide of E. coli and S. minnesota LPS and causes macroagglutination of bacteria expressing rough LPS, our experiments have shown that SP-D can enhance the phagocytosis of smooth and rough nonmucoid strains, but more selectively modifies the cellular responses to rough P. aeruginosa LPS. Thus, SP-D may play particularly important roles in the host response to nonmucoid strains of P. aeruginosa. An important implication of our findings is that the outcome of future in vivo studies of bacterial and LPS challenge could be highly dependent on bacterial strain and LPS structure.


   Acknowledgments
 
The authors thank Prof. H. Haagsman, Dr. W. Steinhilber, and Prof. Dr. K. Schäfer for providing purified SP-A, and Prof. H.-W. L. Ziegler-Heitbrock for the Mono Mac 6 cell line. They also thank Yvonne Wüst for excellent technical assistance. This paper includes part of the thesis of B.S. and D.S., and was supported by the Ludwig-Maximilians-Universität München FöFoLe and the Mukoviszidose e.V.

Received in original form May 14, 2002

Received in final form September 17, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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