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Am J Physiol Lung Cell Mol Physiol 281: L776-L785, 2001;
1040-0605/01 $5.00
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Vol. 281, Issue 4, L776-L785, October 2001

Interaction of bacterial lipopolysaccharide with mouse surfactant protein C inserted into lipid vesicles

Luis Augusto1, Karine Le Blay1, Genevieve Auger2, Didier Blanot2, and Richard Chaby1

1 Endotoxin Group and 2 Laboratory of Bacterial Envelopes and Antibiotics, Unité Mixte de Recherche-8619 of the National Center for Scientific Research, University of Paris-Sud, 91405 Orsay, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Infection of the respiratory tract is a frequent cause of lung pathologies, morbidity, and death. When bacterial endotoxin [lipopolysaccharide (LPS)] reaches the alveolar spaces, it encounters the lipid-rich surfactant that covers the epithelium. Although binding of hydrophilic surfactant protein (SP) A and SP-D with LPS has been established, nothing has been reported to date on possible cross talks between LPS and hydrophobic SP-B and SP-C. We designed a new binding technique based on the incorporation of surfactant components to lipid vesicles and the separation of unbound from vesicle-bound LPS on a density gradient. We found that among the different hydrophobic components of mouse surfactant separated by gel filtration or reverse-phase HPLC, only SP-C exhibited the capacity to bind to a tritium-labeled LPS. The binding of LPS to vesicles containing SP-C was saturable, temperature dependent, related to the concentrations of SP-C and LPS, and inhibitable by distinct unlabeled LPSs. Unlike SP-A and SP-D, the binding of SP-C to LPS did not require calcium ions. This LPS binding capacity of SP-C may represent another antibacterial defense mechanism of the lung.

endotoxin; surfactant protein A; surfactant protein B; surfactant protein D


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PENETRATION OF MICROORGANISMS into the body via the airways represents one of the major routes of infection. This potential risk is increased in patients requiring long-term ventilation or airway stenting and leads to a high frequency of nosocomial infections. Because the lung is constantly exposed to infectious challenges, it depends on a complex system of defense mechanisms to facilitate the clearance of pathogens and prevent the development of infections. Airway secretions actively participate to this defense system. Various components such as mucins, antibacterial agents, antioxidants, and antiproteases contribute to respiratory epithelium protection. One of these secreted materials is a surface tension-lowering agent termed pulmonary surfactant that is synthesized by alveolar type II cells (18). This material is a mixture of lipids and proteins. Four major surfactant proteins (SPs) have been described to date: two of these (SP-A and SP-D) are hydrophilic and belong to the C-type (collagen-like) mammalian lectin family referred to as collectins (5), whereas the other two (SP-B and SP-C) are hydrophobic. SPs play roles in recycling the surfactant back to the epithelium (46), in regulating its exocytosis from the alveolar cells (47), and in modulating host defense functions in the lung (32, 43).

Bacterial components, particularly lipopolysaccharide (LPS), can induce lung injury and acute respiratory distress syndrome (ARDS) (34, 41). A surfactant dysfunction contributes, to a large extent, to this pathology (13). Because surfactant replacement has been shown to improve pulmonary function in endotoxin-induced lung injury (28) and because pulmonary surfactant also displays host defense capacities unrelated to its surface tension-lowering activity (32), it appeared of interest to search for possible cross talks between surfactant and LPS. Among the four major SPs identified so far, two of these, SP-A and SP-D, have already been shown to interact with LPS of various phenotypes (22, 27). Although SP-A is a carbohydrate binding protein (17), it may interact with the lipid A moiety of LPS (42), whereas SP-D may interact with the inner core oligosaccharide region of LPS (25). The collagenous domain of these collectins is the ligand for receptors on phagocytes, and their lectin domain recognizes bacterial and viral oligosaccharides (8, 39). In contrast, nothing has been reported on the possible interactions of the hydrophobic SPs (SP-B and SP-C) with LPS. The aim of this study was to determine whether such interactions can occur.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The LPSs from Salmonella minnesota (rough mutant Re 595) and Escherichia coli (serotype 0127:B8) were from Sigma (St. Louis, MO). The LPS from Bordetella pertussis (vaccinal strain 1414) was prepared in our laboratory as previously described (26). Porcine SP-C was provided by Dr. Jan Johansson (Karolinska Institutet, Stockholm, Sweden). The tripalmitoyl pentapeptide was from Bachem (Bubendorf, Switzerland). Linoleoyl-palmitoyl-L-alpha -phosphatidylinositol, dipalmitoyl-L-alpha -phosphatidyl-DL-glycerol, L-alpha -phosphatidylcholine (type XV-E from egg yolk), N-palmitoyl-D-sphingomyelin, N-palmitoyl-D-sphingosine, n-octyl-beta -D-glucopyranoside, bovine albumin, and fluorescamine were from Sigma. Ovalbumin was from Miles Laboratories (Elkhart, IN). All HPLC solvents (LiChrosolv grade) were from Merck (Darmstadt, Germany). Tritium-labeled borohydride (481 GBq/mmol) was from Amersham Pharmacia Biotech. Tritium-labeled choline (2.8 TBq/mmol) was from NEN (Boston, MA). After biosynthetic labeling of the mouse lung adenocarcinoma cell line MLE-12 with tritium-labeled choline, tritium-labeled phosphatidylcholine ([3H]PC) was extracted from the cells with a chloroform-methanol-water mixture (10:5:3 by volume). The purity of [3H]PC recovered in the organic phase was assessed by the presence of a single radioactive band (migration referred to solvent front = 0.64) detectable by thin-layer chromatography (TLC) on Silica gel 60 plates (Merck) developed in isobutyric acid-1 M ammonium hydroxide (5:3). The liquid scintillation reagents Aqualyte and Lipofluor were from Baker (Deventer, The Netherlands).

Preparation of tritium-labeled LPS. The labeling was done by a modification of the procedure of Watson and Riblet (45). A sample (2 mg) of LPS from S. minnesota Re 595 was incubated for 150 min at room temperature in 0.5 ml of sodium periodate (3 × 10-2 M) and reincubated for 30 min after the addition of 15 µl of 1 M ethylene glycol. The material was lyophilized, and the salts were removed by two centrifugations (15 min at 100,000 g) after resuspension in 200 µl of water. After resuspension of the pellet in 200 µl of ice-cold borate buffer (0.05 M, pH 9.5), 100 µl of an ice-cold solution of NaB 3H4 in the same buffer (0.46 GBq, 481 GBq/mmol) were added, and the suspension was maintained for 18 h at 4°C with magnetic stirring. Excess sodium borohydride was destroyed with 5 µl of acetic acid, and the salts were removed by two centrifugations (15 min at 100,000 g) after resuspension in 400 µl of an ice-cold water-ethanol mixture (1:1 by volume). The radiolabeled LPS was solubilized by a modification of the method of Shands and Chun (38). Briefly, the radioactive pellet was suspended in 500 µl of a 0.05 M Tris · HCl-0.01 M EDTA buffer (pH 7), and the mixture was maintained for 1 min at pH 4 by the addition at 4°C, with stirring, of 11 µl of 0.5 M HCl. The mixture was then readjusted to pH 7 with 5.5 µl of 1 M NaOH, dialyzed for 2 h against the Tris · HCl-EDTA buffer and for 2 h against distilled water, and lyophilized. The specific activity of the radiolabeled LPS was 9 × 105 counts · min-1 (cpm) · µg-1 (2 × 103 cpm/pmol).

To assess that the bioactivity of the LPS was not destroyed during the radiolabeling procedure, we analyzed the ability of [3H]LPS to induce nitric oxide production in the mouse monocyte/macrophage cell line RAW 264.7. After incubation for 30 min with mouse interferon-gamma (5 U/ml), the cells were exposed (24 h at 37°C) to different concentrations of unlabeled or tritium-labeled LPS Re 595. Fifty microliters of the culture supernatant were then incubated with 100 µl of Griess reagent (equal volumes of 1% sulfanilamide in 1 M HCl and 0.1% N-1-naphthylethylenediamine in water). After 30 min at room temperature in the dark, the optical densities were measured on a Dynatech ELISA plate reader at 570 nm. NO<UP><SUB>2</SUB><SUP>−</SUP></UP> concentrations were calculated by comparison with a standard curve obtained with NaNO2 dissolved in culture medium. We found that nitric oxide (NO) production induced by 2 and 5 ng/ml of [3H]LPS (32 ± 2 and 53 ± 3 nmol/ml of NO, respectively) was not significantly different from that induced by the same concentrations of unlabeled LPS (30 ± 1 and 51 ± 3 nmol/ml of NO, respectively). This result was taken as a good indication that the bioactivity of LPS had not been modified by the radiolabeling procedure.

Preparation of surfactant components. Crude surfactant was isolated from the bronchoalveolar lavage fluid of 5- to 10-wk-old Swiss mice (Janvier, Le Genest Saint-Isle, France) on a NaCl-NaBr density gradient as described by Katyal et al. (24). The hydrophobic constituents (fraction containing SP-B, SP-C, and phospholipids) were separated from the hydrophilic constituents (fraction containing SP-A and SP-D) by extraction (1 h at 4°C) in a mixture of chloroform-methanol-1 M HCl (60:40:0.1 by volume) and centrifugation (10 min at 12,000 g). Extraction (1 h at 4°C) of the hydrophobic material with a mixture of ethanol-diethylether (1:3 by volume) and centrifugation (15 min at 12,000 g) according to the method of Beers et al. (1) allowed the separation of SP-B (in the pellet) from SP-C and phospholipids (in the supernatant). The presence of SPs in different fractions was assessed by silver nitrate staining of tricine-SDS-PAGE gels (35) for SP-B and SP-C. PC, the major phospholipid of the surfactant preparation, was detected by TLC on silica gel 60 plates developed in a hexane-chloroform-methanol mixture (5:1:1 by volume) and was revealed by charring with sulfuric acid (10% by volume in ethanol).

Purification of hydrophobic components by gel chromatography. Hydrophobic constituents extracted from mouse surfactant were purified by sequential gel filtration on columns (750 × 17 mm) of Sephadex LH-20 and LH-60 (Pharmacia Biotech) with the solvent system of chloroform-methanol-0.1 M HCl (10:10:0.5 by volume) at a flow rate of 0.4 ml/min as described by Pérez-Gil et al. (31). The eluted material was detected by its absorbance at 240 nm and by its fluorescence after derivatization of the amine functions with fluorescamine (40).

Reverse-phase HPLC. Before analysis by HPLC, the hydrophilic components and phospholipids were removed with a modification of the method of Beers et al. (1). Briefly, crude surfactant obtained by density gradient (2.5 mg) was sonicated in a solution of 5 mM Tris · HCl and 75 mM NaCl (pH 7.4). A mixture (2.5 ml) of diisopropylether-1-butanol (3:2 by volume) was then added. After being stirred, the hydrophobic surfactant components were isolated at the interphase (hydrophilic components were in the aqueous phase and phospholipids in the organic phase). The first interphase was reextracted twice with the organic solvent and once more with the aqueous buffer. The material in the interphase (devoid of phospholipids as assessed by TLC analysis) was recovered by evaporation of the solvent under a nitrogen stream. A fraction of this hydrophobic material (5 mouse equivalents) was analyzed by reverse-phase HPLC. The sample was dissolved in 100 µl of solvent A (0.2% trifluoroacetic acid and 75% methanol in water) and applied on a C18 column from Waters (µBondapak C18, 10 µm, 300 × 3.9 mm). Analysis was carried out at a flow rate of 0.7 ml/min, first with solvent A for 10 min and then with a linear gradient (2.5%/min for 30 min) of solvent B (0.1% trifluoroacetic acid in 2-propanol) in solvent A. Absorbance of the effluent was monitored at 225 nm.

Amino acid analysis. A sample of mouse surfactant component purified by HPLC (4 mouse equivalents) was taken up in 200 µl of 6 M HCl and hydrolyzed at 105°C for 72 h. Amino acid analyses were performed on a Biotronik LC 2000 analyzer equipped with a Dionex DC6A resin column (Dionex, Sunnyvale, CA) and a Spectra-Glo fluorometer (Gilson, Villiers-le-Bel, France). The postcolumn detection was carried out by measuring the fluorescence intensity of isoindole derivatives obtained by the action of o-phthalaldehyde in the presence of 2-mercaptoethanol (2, 33). Amino acid standard H from Pierce (Rockford, IL), hydrolyzed under the same conditions, was used as the calibration mixture. The results were computed with a D-7500 integrator (Merck-Hitachi, Fontenay-sous-Bois, France). Five amino acid residues, Val, Leu, Ile, Phe, and His [theoretical occurrence of 12, 7, 3, 1, and 1 residues/molecule of mouse SP-C, respectively (12)], were used for quantitation of mouse SP-C in the sample.

LPS binding assay. Glass tubes containing PC (0.3 mg) alone or mixed with the hydrophobic material to be tested were evaporated to dryness. After sonication with a solution of BSA (60 µl, 1 mg/ml in 0.15 M NaCl), [3H]LPS (3.6 × 105 cpm; 290 µl in 0.15 M NaCl) was added, and the mixture was incubated (2 h at 20°C) with gentle rotation. The radioactive mixture was adjusted to 1.185 g/ml by the addition of 350 µl of a solution of 1.1% NaCl and 46% NaBr. A discontinuous gradient was prepared by the sequential addition of 23% NaCl (0.7 ml), the radioactive suspension (0.7 ml), 20.5% NaCl (0.7 ml), 16.5% NaCl (0.7 ml), and 8% NaCl (0.2 ml). After centrifugation (65,000 g for 90 min), the radioactivity of the fractions collected from the top was determined by liquid scintillation and is expressed as a percentage of the total radioactivity recovered.

Data processing. In some experiments (see Figs. 2 and 6), data were fitted to a four-parameter logistic (sigmoid) curve with the Marquardt-Levenberg curve-fitting algorithm provided in the SigmaPlot 2000 program (SPSS, Chicago, IL).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Design of an LPS binding assay adapted to hydrophobic surfactant components. Several cell surface molecules (membrane CD14, scavenger receptor, beta 2-integrins) as well as soluble molecules secreted by cells or shed from their surface [LPS binding protein (LBP), soluble CD14] have been reported to bind LPS (10). Concerning soluble molecules, assays designed to assess their LPS binding capacity consist generally in the attachment of one of the interacting partners (the protein or the LPS) to the surface of beads or plastic wells followed by the estimation of the binding of the second partner (often labeled) to this surface. However, because the biologically active moiety of LPS is the hydrophobic (lipid A) region, the spatial conformation of this region must be preserved in binding assays. An important drawback of the binding technique mentioned above is that the conformation of the hydrophobic region of one of the partners (protein or LPS) is markedly modified by its interaction with the beads or the plastic surface. This is particularly dramatic when the protein that is supposed to bind LPS is itself hydrophobic. Therefore, to analyze interactions between LPS and hydrophobic molecules such as those present in lung surfactant, another binding assay is required.

Because lung surfactant is made of lipoprotein particles consisting of a mixture of proteins (essentially SP-A, SP-B, SP-C, and SP-D) and phospholipids (essentially dipalmitoylphosphatidylcholine) (18) and because these particles can be isolated on appropriate density gradients (24), we examined whether the binding of radiolabeled LPS to vesicles containing surfactant components could be detected after separation of unbound ligand on a density gradient.

Crude surfactant was recovered from mouse bronchoalveolar lavage fluid by the NaCl-NaBr gradient procedure of Katyal et al. (24). The hydrophobic and hydrophilic components were separated by stirring 2.5 mg of this lyophilized material for 1 h at 4°C in 2.5 ml of a mixture of chloroform-methanol-1 M HCl (60:40:0.1 by volume). Insoluble, hydrophilic components were recovered in the pellet after centrifugation for 10 min at 12,000 g. Hydrophobic components were recovered from the supernatant after evaporation of the organic solvents under a nitrogen stream. Vesicles obtained after sonication of mixtures of PC-BSA in the absence and presence of hydrophobic or hydrophilic components of mouse surfactant (2 mouse equivalents) were incubated at 20°C with [3H]LPS. After 2 h, the radioactive mixture was adjusted to 1.185 g/ml, and a discontinuous density gradient was prepared by the sequential addition of solutions of decreasing densities (Fig. 1). After centrifugation (90 min at 65,000 g), the radioactivity of the fractions (50 µl) collected from the top was measured and is expressed as a percentage of total radioactivity recovered. The results in Fig. 1 show that in the absence of surfactant components (in the presence of PC-BSA alone), the radiolabeled LPS was quantitatively recovered in the last (bottom) 10 fractions of the density gradient (A). A similar result was obtained in the presence of two mouse equivalents of the hydrophilic components (Fig. 1C). In contrast, in the presence of two mouse equivalents of the hydrophobic components, almost all of the radiolabeled LPS was found in the first (top) 20 fractions of the density gradient (Fig. 1B). This indicates that at least one of the hydrophobic components of mouse surfactant (consisting essentially of SP-B, SP-C, and phospholipids) binds efficiently to [3H]LPS and traps it on the phospholipid vesicles floating on the top of the density gradient. To confirm that the vesicles of PC are actually localized at the top of the gradient, a small amount (0.4 ng) of [3H]PC (5 × 104 cpm) was added to the unlabeled PC before the gradient separation. The percentage of [3H]PC in the top region (1 ml) of the gradient was determined. We found that with PC-BSA alone and PC-BSA in the presence of SP-C, a large majority of [3H]PC (84.9 ± 4.3 and 86.5 ± 15.2%, respectively) was indeed present in the top region of the gradient.


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  		Fig. 1.   Binding of [3H]lipopolysaccharide (LPS) to surfactant components. Tubes containing phosphatidylcholine (PC; 0.3 mg) alone (A) or added to the hydrophobic (B) or hydrophilic (C) components of mouse surfactant (0.1 mouse equivalent) were evaporated to dryness. After sonication with a solution of BSA (60 µl, 1 mg/ml in 0.15 M NaCl), [3H]LPS [3.6 × 105 counts/min (cpm); 290 µl in 0.15 M NaCl] was added, and the mixture was incubated (2 h at 20°C) with gentle rotation. The density of the radioactive mixture was then adjusted to 1.185 g/ml by addition of 350 µl of a solution of 1.1% NaCl and 46% NaBr. A discontinuous gradient was prepared by sequential addition of 23% NaCl (0.7 ml), the radioactive suspension (0.7 ml), 20.5% NaCl (0.7 ml), 16.5% NaCl (0.7 ml), and 8% NaCl (0.2 ml). After centrifugation (90 min at 65,000 g), the radioactivities of fractions (50 µl) collected from the top were determined by liquid scintillation and are expressed as percentages of the total radioactivity recovered.

Analysis of the LPS binding capacity as a function of the amount of hydrophobic material used indicated a sigmoidal dose-effect relationship (Fig. 2). Fifty percent of the optimal binding was obtained with 0.02 mouse equivalent of the hydrophobic constituents of lung surfactant. Below, a LPS binding index (LBI) is used. The LBI of a sample of surfactant material is defined as the difference between the percentage of [3H]LPS recovered at the top (1 ml) of the density gradient in the presence (total binding) and absence (nonspecific binding) of the sample in the vesicles: LBI (%) = total binding (%) - nonspecific binding (%).


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  		Fig. 2.   Concentration-related LPS binding capacity of the hydrophobic fraction of mouse surfactant. Various amounts of the hydrophobic fraction of mouse surfactant were analyzed for their LPS binding capacity as described in Fig. 1. Results are means ± SD of duplicate experiments. Data are fitted on a 4-parameter logistic (sigmoid) curve.

Analysis of fractions enriched in SP-B and SP-C. For rapid enrichment in SP-B and SP-C, we first used a modification of the extraction procedure of Beers et al. (1). Briefly, the hydrophobic material extracted from 2.5 mg of crude mouse surfactant was stirred for 1 h at 4°C in 2.5 ml of a mixture of ethanol-diethylether (1:3, by volume). After centrifugation for 15 min at 12,000 g, the pellet was reextracted (twice) with the same mixture. The final pellet was considered the SP-B-enriched extract, and the pooled supernatants represented the SP-C-enriched extract (which also contained the phospholipid constituents of the surfactant). Analysis of the LPS binding capacities of the SP-B- and SP-C-enriched extracts (0.5 mouse equivalent) gave a LBI of 13.3% for the former and 73.6% for the latter (subtracted background with PC-BSA alone 9.5%).

A better separation of SP-B and SP-C was performed by sequential chromatography on Sephadex LH-20 and LH-60 as described by Pérez-Gil et al. (31). After removal of phospholipids from the hydrophobic material extracted from 47 Swiss mice by a first chromatography on a Sephadex LH-20 column, the phospholipid-depleted material was submitted to a second chromatography (Fig. 3A) on a Sephadex LH-60 column. The results in Fig. 3A show that two main peaks were detected (fractions 18-35 and 60-79) that, according to literature data (31), should correspond to SP-B and SP-C, respectively. Two minor peaks (fractions 45-59 and 80-90) were also detected.


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  		Fig. 3.   Gel chromatography of the hydrophobic constituents of mouse surfactant. After removal of phospholipids from the hydrophobic material extracted from 47 Swiss mice by a 1st chromatography on a Sephadex LH-20 column, the phospholipid-depleted material was chromatographed on a Sephadex LH-60 column (750 × 17 mm) eluted at a flow rate of 0.4 ml/min with a chloroform-methanol-0.1 M HCl mixture (10:10:0.5 by volume). A: eluted material was detected by its absorbance at 240 nm and by its fluorescence after treatment with fluorescamine. SP, surfactant protein. B: fractions were also analyzed for LPS binding capacity as described in Fig. 1.

Fractions (40 µl) of the effluent were evaporated to dryness, and the LPS binding capacity of the corresponding material was analyzed with [3H]LPS as described above. The results (Fig. 3B) indicated that compound of the first major peak (SP-B) did not bind LPS, whereas the second one did. Some LPS binding activity was also detectable in the last fractions eluted from the column (fractions 75-90). These results suggest that SP-C or compounds with a similar or lower molecular weight can bind LPS.

LPS binding capacity of fractions purified by HPLC. Because a more accurate analysis of the hydrophobic material appeared essential, we used reverse-phase HPLC. Crude surfactant isolated from 40 Swiss mice (114 mg) was submitted three times for 1 h at 4°C to the extraction procedure with diisopropylether-1-butanol (20 ml) as described by Beers et al. (1). A material consisting of hydrophobic surfactant constituents devoid of PC was recovered at the interphase. A fraction of this material (corresponding to an extract from 5 mice) was analyzed by reverse-phase HPLC on a C18 column. Figure 4 shows that nine main peaks were detected by this method. Fractions (200 µl) of these peaks were evaporated to dryness, and the LPS binding capacity of the corresponding material was analyzed with [3H]LPS. The results in Table 1 clearly show that the compound present in peak 7 is the only one that binds to LPS.


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  		Fig. 4.   HPLC analysis of the hydrophobic constituents (nos. above peaks) of mouse surfactant. After removal of phospholipids by extraction with a mixture of diisopropylether-1-butanol (3:2 by volume), the hydrophobic material (5 mouse equivalents) was dissolved in 100 µl of solvent A (0.2% trifluoroacetic acid and 75% methanol in water) and analyzed by reverse-phase HPLC on a µBondapak C18 column (300 × 3.9 mm), first with solvent A for 10 min and then with a linear gradient (2.5%/min for 30 min) of solvent B (0.1% trifluoroacetic acid in 2-propanol) in solvent A at a flow rate of 0.7 ml/min. Absorbance of the effluent was monitored at 225 nm. Standards of lauric (C12), myristic (C14), palmitic (C16), stearic (C18), and arachidic (C20) acids and a sample of porcine SP-C were also analyzed by HPLC under the same conditions.


                              
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  		Table 1.   LBI of HPLC fractions of hydrophobic components of mouse surfactant

Presence of covalently linked palmitoyl chains in component 7 isolated from HPLC. One specific feature of SP-C is that this surfactant component contains two palmitoyl chains covalently attached via S-ester bonds to cysteine residues of the polypeptide chain. These S-ester bonds can be easily cleaved by mild alkaline treatment (36). On the other hand, it has been reported that an isoform of pig SP-C contains a third palmitoyl moiety linked to the epsilon -amino group of lysine-11 via a chemically stable amide bond (16). Furthermore, in many palmitoylated and myristoylated peptides and proteins, the fatty acid chain is linked to an amino group via an amide bond (21).

The cleavage of these amide bonds requires drastic treatments such as strong acid hydrolysis. To determine whether covalently linked fatty acids are present in the HPLC component 7, one mouse equivalent of this fraction was submitted either to a strong acid hydrolysis (4 M HCl for 2 h at 100°C) or to a mild alkaline treatment (0.7 M NH4OH for 3 h at 37°C). In the latter case, the pH was adjusted to 2 after the alkaline treatment to convert water-soluble carboxylates to chloroform-soluble carboxylic acids. The HCl and NH4OH hydrolysates were then extracted with chloroform, methylated with diazomethane, and analyzed by gas-liquid chromatography coupled to mass spectrometry. The results (Fig. 5) show that in the two hydrolysates, only one fatty acid methyl ester was detectable by gas-liquid chromatography, with a retention time (18.86 min) corresponding to that of a methyl palmitate reference. The mass spectra of the compounds with this retention time exhibit a molecular ion at a mass-to-charge ratio (m/z) of 270.4 (33.21% of base peak) and main ions at m/z of 74.1 (100.00%), 87.1 (91.34%), 143.2 (28.66%), 227.3 (22.50%), 239.3 (13.52%), and 241.3 (5.88%) and are thus identical to the mass spectrum of a methyl palmitate standard.


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  		Fig. 5.   Analysis of the fatty acid residues of component 7 by gas-liquid chromatography-mass spectrometry (GC-MS). After strong acid treatment (4 M HCl, 2 h at 100°C; B) or mild alkaline treatment (0.7 M NH4OH, 3 h at 37°C; C) of the material isolated by HPLC in peak 7, carboxyl groups were methylated and chloroform-soluble compounds were analyzed by GC-MS with a DB5ms capillary column (30 m) coupled to a Finnigan MAT 95S mass spectrometer. A 2-step temperature gradient starting at 130°C (3°C/min for 10 min, followed by 4°C/min for 20 min) was used. Ion mass-to-charge ratio (m/z) of 87 was monitored for specific detection of fatty acid methyl esters. Retention times and mass spectra were compared with those of the methyl esters of lauric (C12), myristic (C14), and palmitic (C16) acids (A).

Methyl palmitate was not detected after direct esterification of the HPLC component 7 without HCl or NH4OH treatment (data not shown). These results indicate that component 7 isolated by HPLC contains a molecule with palmitoyl chain(s) linked via covalent bonds as labile as the S-ester bonds of SP-C. Analyses of the other fractions isolated by HPLC by the same method indicated that component 7 was the only one that contained covalently linked fatty acid chains (data not shown).

Optimal features of SP-C-containing vesicles required for interaction with LPS. The presence of palmitoyl residues in mouse surfactant component 7 strongly suggests that this compound is mouse SP-C. This was confirmed by comparison of the HPLC analysis of this compound with a sample of porcine SP-C (provided by Dr. Jan Johansson). The results (Fig. 4) indicated that the retention time of mouse surfactant component 7 is identical (~28 min) to that of the porcine SP-C standard.

We then determined some of the parameters (amount of SP-C, presence of calcium, temperature) required for optimal binding of LPS to SP-C-containing vesicles. The binding of [3H]LPS (3.6 × 105 cpm) to vesicles of PC-BSA (300 µg/60 µg) containing various amounts of component 7 (0-120 pmol of mouse SP-C according to amino acid estimation) was analyzed after centrifugation on a gradient of NaCl-NaBr as described above. The results (Fig. 6) show that the binding of [3H]LPS to the vesicles is correlated to the content of SP-C. An optimal binding of 140 pmol of [3H]LPS was obtained with vesicles containing 70 pmol of SP-C. Vesicles with higher contents of SP-C did not bind more LPS. This may indicate that vesicles cannot accommodate higher amounts of SP-C on their surface.


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  		Fig. 6.   Binding of radiolabeled LPS to vesicles with increasing amounts of SP-C. Increasing amounts of HPLC-purified mouse SP-C (peak 7) were incorporated into PC-BSA (300 µg/60 µg) vesicles, which were incubated (2 h at 20°C) with 180 pmol of [3H]LPS (3.6 × 105 cpm). The radiolabeled LPS bound to the vesicles was recovered at the top (1 ml) of a NaCl-NaBr gradient as described in Fig. 1. Values obtained with radiolabeled LPS bound to empty vesicles of PC-BSA (11%) were subtracted from the data. Results are means ± SD of duplicate experiments. Data are fitted on a 4-parameter logistic (sigmoid) curve.

The role of BSA in the binding technique was only to reduce nonspecific interactions between [3H]LPS and the lipid vesicles. Indeed, with vesicles devoid of SP-C, we measured a LBI of 9 ± 2% in the presence of BSA and 13 ± 0.5% in the absence of BSA. Therefore, BSA partially reduces nonspecific binding. On the other hand, this particular protein is not critical for the interaction of LPS with SP-C and can be replaced by another protein such as ovalbumin. The LBI obtained in the presence of ovalbumin (74.5 ± 2.3%) was not significantly different from that obtained with BSA (78.2 ± 5.4%).

When experiments were carried out in the presence and absence of 1.5 mM Ca2+, we observed similar binding levels of [3H]LPS (data not shown). On the other hand, when vesicles were exposed to [3H]LPS at 0°C instead of 20°C, the binding of LPS to vesicles containing SP-C was considerably reduced compared with the nonspecific binding on vesicles without SP-C (Fig. 7). It appears, therefore, that the interaction between SP-C and LPS is calcium independent but is markedly temperature dependent.


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  		Fig. 7.   Influence of the temperature on the binding of [3H]LPS. Vesicles of PC-BSA (300 µg/60 µg) made up without and with 6 pmol of HPLC-purified mouse SP-C (peak 7) were incubated for 2 h at 20°C or at 0°C with 20 µg/ml of [3H]LPS (6.3 × 106 cpm). The radiolabeled LPS bound to the vesicles was recovered at the top (1 ml) of a NaCl-NaBr gradient carried out at room temperature as described in Fig. 1.

Specificity of the interaction between mouse SP-C and LPS. Incubation of vesicles with increasing amounts of radiolabeled LPS clearly indicates that the binding is saturable (Fig. 8A). When vesicles containing SP-C were preincubated with unlabeled LPS (up to 1 mg/ml) 2 h before the addition of [3H]LPS, a complete inhibition of the binding of the radiolabeled LPS was observed (Fig. 8B), thus indicating that the binding of LPS to SP-C is specific.


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  		Fig. 8.   Saturation and inhibition of the interaction between LPS and SP-C. Vesicles of PC-BSA (30 µg/6 µg) containing 6 pmol of HPLC-purified mouse SP-C (peak 7) were incubated (2 h at 20°C) with increasing concentrations of [3H]LPS (A) or were preincubated with increasing concentrations of the homologous unlabeled LPS (2 h at 20°C) and reincubated (2 h at 20°C) after addition of 1.1 µg/ml (3.6 × 104 cpm) of radiolabeled LPS (B). The radiolabeled LPS bound to the vesicles was recovered at the top (1 ml) of a NaCl-NaBr gradient as described in Fig. 1. Values obtained with radiolabeled LPS bound to empty vesicles of PC-BSA were subtracted from the data. Results are means ± SD of duplicate experiments.

To examine the possible influence of high concentrations of unlabeled LPS on the density of the lipid vesicles, we used tritium-labeled vesicles obtained by incorporation of [3H]PC to the unlabeled PC. Without LPS, 84.9 ± 4.3% of the radioactivity (and thus of the vesicles) was found in the floating (top 1-ml) region of the gradient. In the presence of high amounts of LPS (500 µg/ml), 74.1 ± 6.7% of [3H]PC was found in the same region. Therefore, high amounts of LPS induced a small (12.7%) decrease in the amount of vesicles present in the floating region of the gradient. However, this moderate effect cannot account for the considerable (86.7%) inhibition of the binding of radiolabeled LPS observed in Fig. 8A.

To assess the specificity of the interaction concerning SP-C, it was important to show that other hydrophobic compounds unrelated to SP-C do not exhibit similar LPS binding features when incorporated into PC-BSA vesicles. We used one compound with an eight-carbon aliphatic chain (n-octyl-beta -D-glucopyranoside), two compounds containing one palmitoyl residue (N-palmitoyl-D-sphingomyelin and N-palmitoyl-D-sphingosine), one compound with two different fatty acid chains (linoleoyl-palmitoyl-L-alpha -phosphatidylinositol), one compound with two palmitoyl chains (dipalmitoyl-L-alpha -phosphatidyl-DL-glycerol), and one compound with three palmitoyl chains (tripalmitoyl pentapeptide). The six compounds as well as mouse SP-C were used at the same concentration (200 pmol in vesicles consisting of 300 µg of PC and 60 µg of BSA). The results in Table 2 show that LPS binding capacity was exhibited exclusively by SP-C, with the six other compounds being inactive.

                              
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  		Table 2.   LPS binding index of mouse SP-C and other lipids

The last question was to assess that the S. minnesota LPS used above is not unique in its capacity to interact with mouse SP-C. We then used the technique of competition with unlabeled LPS described in Fig. 8B. We found (Fig. 9) that two other unlabeled LPSs (from B. pertussis and E. coli) inhibited the binding of [3H]LPS as efficiently (89 ± 3 and 88 ± 3% of inhibition, respectively) as the homologous unlabeled LPS from S. minnesota Re 595 (80 ± 3% of inhibition). This means that LPSs of different bacterial origins can interact with mouse SP-C.


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  		Fig. 9.   Inhibition of the binding of [3H]LPS with different unlabeled LPSs. Vesicles of PC-BSA (30 µg/6 µg) containing 6 pmol of HPLC-purified mouse SP-C (peak 7) were preincubated (2 h at 20°C) with 714 µg/ml of LPSs from Salmonella minnesota (mutant Re 595), Bordetella pertussis (strain 1414), and Escherichia coli (serotype 0127:B8) or with saline alone. The vesicles were then reincubated (2 h at 20°C) after addition of 21 µg/ml of [3H]LPS (6.7 × 105 cpm). The radiolabeled LPS bound to the vesicles was recovered at the top (1 ml) of a NaCl-NaBr gradient as described in Fig. 1. The value obtained with radiolabeled LPS bound to empty vesicles of PC-BSA (1.6 × 104 cpm) was subtracted from the data. Results are means ± SD of triplicate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The lung is constantly exposed to a vast array of particulate matter including environmental bacteria and commensal microorganisms of the oropharyngeal flora. Some bacterial constituents, particularly endotoxin (LPS) of gram-negative bacteria, are known to induce deleterious effects in the host (23, 30). Nevertheless, humans and animals are generally protected against bacteria and their products by two barriers: the mucociliary system that lines the conducting airways and removes aspirated particles and the innate immune system of the alveolar space that detects bacterial products and attracts phagocytic leukocytes. The latter is an efficient defense mechanism, and its recognition function is served, in part, by soluble molecules secreted by the different cell types present in the alveolar space, including neutrophils, macrophages, and type II epithelial cells. For example, it has been shown that epithelial type II cells can produce lysozyme, defensins (37), antimicrobial peptides (4, 44), and LBP (7). Epithelial type II cells are also known as a source of pulmonary surfactant (18), and it has been already established that the two main hydrophilic constituents of this surfactant, SP-A and SP-D, can interact with LPS (22, 27). The results of our study indicate that another compound produced by epithelial type II cells, the hydrophobic surfactant component SP-C, interacts with LPS.

It should be noted that in our binding assay, we measured the interaction of LPS with vesicles containing SP-C and PC. Therefore, the possibility that LPS binds to PC more avidly in the presence of SP-C cannot be completely excluded. This alternative explanation of the binding does not actually affect the global finding of this study inasmuch as SP-C is always associated with PC in physiological conditions.

Our study is the first report on an interaction of LPS with a hydrophobic surfactant component. However, this finding is not completely surprising and could have been expected because there are some indications that in tissues other than the lung, some hydrophobic molecules can bind to LPS. For example, it has been reported that LPS binds to lipoproteins of high and low density (9, 11) and to the glycolipid asialo-GM1 (15). More recently, a binding between certain lipopolyamines and LPS has been observed (3).

It should be noted that the binding technique designed in our study is restricted to molecules that insert efficiently into the outer layer of PC-BSA vesicles. The hydrophilic components of mouse surfactant do not apparently fulfill this condition because, according to our data (Fig. 1), they do not allow the binding of LPS to the vesicles. Therefore, despite a clearly established capacity to bind LPS (22, 27), SP-A and SP-D were ineffective in our vesicle-based LPS binding assay. We cannot exclude, however, that in our experiments, the observed inability of the hydrophobic surfactant components to bind LPS could be due to some denaturation of these proteins during the extraction procedure with chloroform and methanol. Another marked difference between the binding features of LPS with SP-C versus SP-A or SP-D concerns the requirement for calcium. SP-A and SP-D belong to a group of collagen-like calcium-dependent lectins called collectins. The binding to LPS via their carbohydrate recognition domain requires calcium (42). In contrast, we found that the binding of SP-C to LPS is calcium independent (data not shown). On the other hand, a marked temperature dependence between LPS and SP-C was observed, the binding being almost completely abolished at 0°C (Fig. 7). Therefore, SP-C is likely to represent a complementary tool used by the innate immune system in the lung, which works under conditions at which SP-A and SP-C are not fully operative.

With respect to the physiological role of the interaction between SP-C and LPS, two possible mechanisms similar to those demonstrated in blood circulation can be proposed. The first mechanism could be a scavenging role of SP-C similar to that of plasma high-density lipoproteins (HDLs). It has been established that after presentation by LBP (6) or phospholipid transfer protein (20), LPS binds to HDLs and becomes unable to induce tumor necrosis factor-alpha production in macrophages (14) but is cleared by phagocytic cells bearing HDL receptors (29). A similar mechanism of neutralization and/or clearance of LPS in the lung via SP-C is thus conceivable, although cellular receptors for SP-C have not yet been reported. The second hypothesis to be considered for a physiological role of the LPS-SP-C interaction could be an enhancement of LPS effects similar to those induced by LBP or soluble CD14. On this assumption, SP-C can play the role of a shuttle by presenting LPS to an appropriate signaling LPS receptor of lung cells.

The two assumptions proposed above being antinomic (neutralization vs. enhancement of LPS effects), further studies are required to determine which of these actually occurs in vivo. Such studies would be of particular importance insofar as trials for the clinical applications of SP-C are presently undertaken, showing that SP-C improved oxygenation in some models of ARDS (19, 28). A better understanding of the physiological role of the interaction between LPS and SP-C will certainly help determine whether this type of surfactant replacement therapy is advisable or inadvisable in lung pathologies such as bacterial pneumonia and sepsis-induced ARDS in which endotoxins take part.

The second point that deserves close scrutiny is to understand at the molecular level the interaction between LPS and SP-C. Experiments designed to examine the contribution of the different regions of the two molecules in their binding are in progress.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Jan Johansson (Karolinska Institutet, Stockholm, Sweden) for providing porcine surfactant protein C. We also thank Félix Perez for gas chromatography-mass spectrometry analyses and Philippe Minard and Monique Synguelakis for assistance.


    FOOTNOTES

This work was supported by a grant from the Direction des Systèmes de Forces et de la Prospective (contract 99.34.033).

Address for reprint requests and other correspondence: R. Chaby, Equipe "Endotoxines," UMR-8619 du C.N.R.S., Bâtiment 430, Université de Paris-Sud, 91405 Orsay, France (E-mail: richard.chaby{at}bbmpc.u-psud.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 20 February 2001; accepted in final form 7 June 2001.


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