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 |
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
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INTRODUCTION |
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.
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METHODS |
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-
-phosphatidylinositol, dipalmitoyl-L-
-phosphatidyl-DL-glycerol,
L-
-phosphatidylcholine (type XV-E from egg yolk),
N-palmitoyl-D-sphingomyelin,
N-palmitoyl-D-sphingosine, n-octyl-
-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-
(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
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 |
Design of an LPS binding assay adapted to hydrophobic surfactant
components.
Several cell surface molecules (membrane CD14, scavenger receptor,
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.
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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.
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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.
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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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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
-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).
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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-
-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-
-phosphatidylinositol), one
compound with two palmitoyl chains
(dipalmitoyl-L-
-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.
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 |
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-
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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