Pulmonary surfactant inhibits LPS-induced nitric oxide
production by alveolar macrophages
P. R.
Miles1,2,
L.
Bowman1,
K. M. K.
Rao1,
J. E.
Baatz3, and
L.
Huffman1,2
1 Health Effects Laboratory
Division, National Institute for Occupational Safety and Health,
Morgantown 26505; 2 Department of
Physiology, West Virginia University School of Medicine, Morgantown,
West Virginia 26506; and
3 Department of Pediatrics,
Medical University of South Carolina, Charleston, South Carolina
29425-3313
 |
ABSTRACT |
The objectives of
this investigation were 1) to report
that pulmonary surfactant inhibits lipopolysaccharide (LPS)-induced nitric oxide (· NO) production by rat alveolar macrophages,
2) to study possible mechanisms for
this effect, and 3) to determine which surfactant component(s) is responsible. · NO produced
by the cells in response to LPS is due to an inducible · NO
synthase (iNOS). Surfactant inhibits LPS-induced · NO
formation in a concentration-dependent manner; · NO
production is inhibited by ~50 and ~75% at surfactant levels
of 100 and 200 µg phospholipid/ml, respectively. The inhibition is
not due to surfactant interference with the interaction of LPS with the
cells or to disruption of the formation of iNOS mRNA. Also, surfactant
does not seem to reduce · NO formation by directly affecting
iNOS activity or by acting as an antioxidant or radical scavenger.
However, in the presence of surfactant, there is an ~80% reduction
in the amount of LPS-induced iNOS protein in the cells. LPS-induced
· NO production is inhibited by Survanta, a surfactant
preparation used in replacement therapy, as well as by natural
surfactant. · NO formation is not affected by the major lipid
components of surfactant or by two surfactant-associated proteins,
surfactant protein (SP) A or SP-C. However, the hydrophobic SP-B
inhibits · NO formation in a concentration-dependent manner; · NO production is inhibited by ~50 and ~90% at SP-B
levels of 1-2 and 10 µg/ml, respectively. These results show
that lung surfactant inhibits LPS-induced · NO production by
alveolar macrophages, that the effect is due to a reduction in iNOS
protein levels, and that the surfactant component responsible for the
reduction is SP-B.
lipopolysaccharide; surfactant protein B; hydrophobic surfactant
proteins; inducible nitric oxide synthase
 |
INTRODUCTION |
NITRIC OXIDE (· NO) is a free radical that is
produced by a variety of cell types in the lungs. The synthesis of
· NO from L-arginine
is catalyzed by a family of enzymes known as · NO synthases (NOSs). One very important isoform of this enzyme is inducible NOS
(iNOS). It has been shown that · NO produced by iNOS plays an
important role in defense against airborne pathogens and is involved in
tissue damage associated with inflammatory processes in the lungs (11,
17). Upregulation of iNOS is transcriptionally regulated and can occur
after exposures to inflammatory stimuli such as cytokines
and/or endotoxin (11). Examples of some lung cells that can
generate · NO by means of iNOS include alveolar type II cells
(14), lung fibroblasts (18), pulmonary arterial smooth muscle cells
(31), and neutrophils (43).
Alveolar macrophages are mobile phagocytic cells located within the
alveolar regions and small airways of the lungs. These cells represent
a primary line of defense against the adverse effects of inhalation of
bacteria and foreign particles. It is well known that alveolar
macrophages can be induced to produce · NO via iNOS. For
example, some investigators (21, 33) have demonstrated that rat
alveolar macrophages produce · NO in response to inflammatory
stimuli or cytokines, e.g., lipopolysaccharide (LPS) or
interferon-
(IFN-
). These inflammatory stimuli act by causing expression of iNOS mRNA (22, 24). In fact, one of the major
sources of · NO produced by iNOS in the lungs during the
inflammatory process is the alveolar macrophage.
Pulmonary surfactant is a complex mixture of phospholipids (PLs),
lipids, and proteins that lines the alveolar regions of the lungs.
Although its major function is to prevent alveolar collapse by lowering
surface tension forces, lung surfactant is also known to have some
effects on alveolar macrophages. For example, Thomassen et al. (39, 40)
have shown that in vitro exposure of human alveolar macrophages to two
surfactant preparations used for replacement therapy, Exosurf and
Survanta, results in suppression of endotoxin-induced production of
cytokines, i.e., tumor necrosis factor-
(TNF-
), interleukin
(IL)-1
, and IL-6. Other investigators have shown that one of the
major surfactant proteins, surfactant protein (SP) A, has effects on
alveolar macrophages, such as inhibiting the release of TNF-
from
LPS-stimulated cells (25), enhancing phagocytosis (41), or stimulating
the secretion of granulocyte-macrophage colony-stimulating factor from
the cells (4). These results demonstrated that lung surfactant can
affect alveolar macrophage function. In addition, surfactant is part of
the normal environment for these cells in vivo. Because alveolar
macrophages represent a major source of · NO production
during inflammatory processes and because surfactant can affect some
responses of these cells, we wondered what effects surfactant has on
cellular · NO formation. Therefore, the objectives of this
investigation were 1) to determine the effects of rat lung surfactant on · NO production by
LPS-stimulated rat alveolar macrophages,
2) to study some possible mechanisms for these effects, and 3) to
determine which surfactant component(s) is responsible for the effects.
 |
METHODS |
Isolation of alveolar macrophages.
Alveolar macrophages were obtained from specific pathogen-free male
Sprague-Dawley rats (225-300 g; Hilltop Laboratories, Scottdale,
PA). The animals were anesthetized with pentobarbital sodium (150 mg/kg
body wt) and exsanguinated by cutting the abdominal aorta. The trachea, heart, and lungs were then removed from the animals intact. Alveolar macrophages were obtained via bronchoalveolar lavage according to the
method of Myrvik et al. (30). The lungs from each animal were lavaged
eight times with 5 ml phosphate-buffered medium (145 mM NaCl, 5 mM KCl,
9.4 mM
Na2HPO4,
and 1.9 mM
NaH2PO4,
pH 7.4)/g lung weight. The cells were separated from the lavage fluid
by centrifugation at 300 g for 5 min
and then washed three times by alternate centrifugation and
resuspension in phosphate-buffered medium. After the washing procedure,
the cells were resuspended in the medium used for cell culture (culture
medium). The culture medium consisted of Eagle's minimal essential
medium (MEM; BioWhittaker, Walkersville, MD) supplemented
with 1 mM glutamine (GIBCO, Life Technologies, Grand Island, NY), 10 mM
HEPES (Sigma, St. Louis, MO), 100 U/ml of penicillin-streptomycin
(GIBCO), 100 µg/ml of kanamycin (GIBCO), and 10% (vol/vol)
heat-inactivated fetal bovine serum (BioWhittaker), pH 7.4. This medium
was made in endotoxin-free water (BioWhittaker) and filtered through a
0.2-µm Nalgene bottle top filter (Sybron, Rochester, NY). The number
of cells in the suspension was determined by using an electronic cell
counter (model ZB, Coulter
Electronics, Hialeah, FL).
Isolation of pulmonary surfactant.
Pulmonary surfactant was obtained from a separate set of rats. The
trachea, heart, and lungs were removed intact from anesthetized animals
as described in Isolation of alveolar
macrophages. A concentrated form of
alveolar lavage material was obtained by bronchoalveolar lavage of the right lung with 5 ml of phosphate-buffered medium followed by lavage of
the left lung with the same 5 ml of fluid. Alveolar macrophages were
removed from the lavage fluid by centrifugation at 300 g for 5 min and then washed three
times by alternate centrifugation and resuspension in
phosphate-buffered medium. All of these washings were spun at 15,000 g for 10 min. The pellet derived from
these washings was added back to the remaining cell-free lavage
materials because a significant amount of surfactant PLs is recovered
in this pellet (28).
Purified lung surfactant was then obtained according to the method of
King and Clements (19). Briefly, the cell-free lavage material was spun
at 100,000 g for 2 h. The resultant
pellet was resuspended in phosphate-buffered medium and applied to a
linear sodium bromide density gradient (density range 1.028-1.100
g/ml). The gradient was then spun at 81,500 g for 15 h in a SW 27 swinging-bucket rotor (Beckman Instruments, Fullerton, CA). The band containing the
surfactant (density 1.050 ± 0.003 g/ml) was removed and spun at
66,000 g for 1 h. This pellet was
washed two times in phosphate-buffered medium and then resuspended in
culture medium for use as the surfactant preparation in the
experiments. In a separate set of experiments, we determined from
osmolarity measurements that most of the sodium bromide was removed
from the lung surfactant during the wash procedure. PLs were measured
as the phosphorus present in the lipid extracts of surfactant (2), and
PL content, in milligrams, was obtained by multiplying the lipid
phosphorus values by 25 (32).
Isolation of SPs. Three different SPs,
SP-A, SP-B, and SP-C, were investigated in this study. SP-A was a gift
from Dr. Jo Rae Wright (Duke University, Durham, NC) and was purified
from bronchoalveolar lavage material from human alveolar proteinosis patients. Because SP-A is a hydrophilic protein, it was dissolved in
culture medium for delivery to the cells. SP-B and SP-C were obtained
according to a method previously published (42). The proteins were
purified from lipid extracts of bovine lung lavage by chromatography on
C-8 silica and Sephadex L-60. Purity of the proteins was determined
with SDS-PAGE (visualized by silver stain), amino acid analysis, and
compositional analysis (42). The SP-B and SP-C concentrations were
determined with the bicinchoninic acid total protein assay (Pierce,
Rockford, IL), with 0.1% SDS in all samples and BSA as the standard.
Because SP-B and SP-C are hydrophobic proteins, they were incorporated
into lipid vesicles for delivery to the cells. The vesicles were
composed of some of the lipid components of surfactant, i.e.,
dipalmitoylphosphatidylcholine (DPPC),
L-
-phosphatidylcholine-
-oleoyl-
-palmitoyl
(PC),
L-
-phosphatidyl-DL-glycerol (PG), and cholesterol. The molar ratio was 10:5:2:3
(DPPC-PC-PG-cholesterol). All lipids were obtained from Sigma. The
vesicles were prepared as described previously (20). Briefly, lipids
and proteins (SP-B or SP-C) were dissolved in ethanol. Liposomes were
formed by injecting the dissolved materials into culture medium warmed
to 48°C. Then the dispersion was sonicated to form vesicles, and
the vesicles were added to the incubation mixtures.
Measurement of · NO
production. Alveolar macrophages suspended in culture
medium were placed into wells of 24-well tissue culture plates (Falcon,
Becton Dickinson, Lincoln Park, NJ). One milliliter of culture medium
containing 2 × 106
cells was placed in each well. The cells were incubated for 22 h at
37°C in an incubator with a humidified atmosphere (relative humidity 90%) of 95% air-5%
CO2. After the incubation period, the supernatants were removed from each well and spun at 13,000 g for 30 s in microfuge tubes to be
certain that all cells were removed. The resultant supernatants were
saved for analysis. The amount of · NO in the supernatants
was measured as the stable oxidation products of · NO,
nitrate and nitrite. All samples were first incubated with
Escherichia coli reductase to convert
the nitrate to nitrite. · NO production was then measured by
using the Greiss reaction (12). The amount of nitrate and nitrite in
the samples was calculated from a standard curve that was constructed from sodium nitrite standards. Conversion of nitrate to nitrite was
checked in each assay with sodium nitrate standards.
The effects of LPS, pulmonary surfactant, and different NOS inhibitors
on · NO production by alveolar macrophages were determined. LPS (from E. coli 026:B6; Difco
Laboratories, Detroit, MI) was added to some wells of the tissue
culture plate so that the final concentration was 10 µg/ml, a
concentration that produces maximal · NO formation. Pulmonary
surfactant, which had been resuspended in tissue culture medium, was
added to some wells so that the final concentration was 25-200
µg PL/ml. Three different NOS inhibitors, NG-monomethyl-L-arginine
acetate (L-NMMA),
NG-nitro-L-arginine
methyl ester (L-NAME), and
aminoguanidine hemisulfate, were included in some wells at a final
concentration of 1 mM. All inhibitors were obtained from RBI (Natick,
MA). In almost all experiments, these substances were added to the
wells before the beginning of the incubation period. However, in some
experiments, some of these substances were added at different times
during the incubation period.
In other experiments, alveolar macrophages were incubated with some of
the lipid and protein components of pulmonary surfactant or with
Survanta, a surfactant preparation used in replacement therapy, in an
attempt to determine which component(s) is responsible for the
surfactant effects. The effects of some of the lipid components of
surfactant, DPPC-PC-PG-cholesterol in a molar ratio of 10:5:2:3, on
LPS-induced · NO production were determined. The lipids were prepared as vesicles as described in Isolation of
SPs. The final amount used was 200 µg PL/ml. In some
experiments, only DPPC, the major component of surfactant, was included
in the incubation medium. DPPC was prepared as vesicles at a final
concentration of 100 µg/ml because it accounts for ~50% of the PLs
in surfactant. In some experiments, Survanta (Ross Laboratories,
Columbus, OH) was added to the incubation mixture at a final
concentration of 200 µg PL/ml. The effects of three SPs, SP-A, SP-B,
and SP-C, on · NO production by alveolar macrophages were
also determined. The proteins were delivered to the cells as described
in Isolation of SPs. None of the
substances used in this study had any effect on the assay for nitrate
and nitrite. Also, by doing cell protein measurements and microscopic
analysis of the supernatants, we determined that none of the substances
affected the ability of the cells to adhere to the culture plates.
Detection of iNOS mRNA. iNOS mRNA
levels were determined in untreated alveolar macrophages and in cells
exposed to LPS alone or to LPS plus lung surfactant. The mRNA levels
were measured by isolating total cellular RNA and then using Northern
blot analysis. The cells were incubated for 22 h in culture
medium alone, in medium with LPS, or in medium with LPS plus lung
surfactant as described in Measurement of
· NO production. After the incubation period,
total cellular RNA was isolated with a guanidinium thiocyanate-based extraction procedure (8) and quantified spectrophotometrically at 260 nm. The relative purities of all RNA samples, as determined by the
ratio of absorbance at 260 nm to that at 280 nm (~1.9), were not
different. The RNA was then size fractioned on a 1.5% agarose gel
containing 2 M formaldehyde and blotted onto a Duralose membrane
(Stratagene, La Jolla, CA) with capillary-mediated bulk flow transfer.
The amount of total RNA analyzed in each sample was 5-10 µg.
iNOS mRNA was indexed by Northern blot analysis with a
32P-labeled cDNA hybridization
probe derived from a plasmid containing a 4,100-bp cDNA fragment for
murine macrophage iNOS (23). The probe was obtained from Drs. C. Lowenstein and S. H. Snyder (Johns Hopkins University. Baltimore, MD).
The cDNA fragment for iNOS was amplified by PCR to produce a
double-stranded cDNA template (GeneAmp DNA Amplification Reagent Kit,
Perkin-Elmer Cetus, Norwalk, CT) with 20-bp synthetic DNA
oligonucleotide primers. The sense primer sequence was
5'-ACTTCCTGGACATTACGACC-3', and the antisense primer
sequence was 5'-CTGCTCCTCGCTCAAGTTCA-3'. A single-stranded DNA hybridization probe was generated by PCR on the double-stranded cDNA template with the antisense primer and
32P-labeled dCTP (ICN
Biochemicals, Costa Mesa, CA). The hybridization probe was purified on
a G-25 Quick Spin column (Boehringer Mannheim, Indianapolis, IN).
Northern blot hybridization was performed with QuickHyb hybridization
buffer (Stratagene) according to the manufacturer's instructions. The
blot was then boiled in RNase-free water for 5 min to remove hybridized
probe, and the amount of 28S rRNA on the blot was determined by using
the hybridization protocol of Barbu and Dautry (1).
To estimate the amount of iNOS mRNA in the alveolar macrophages, the
blots were subjected to image analysis. Images were obtained with a
video camera (model CCD72, Dage-MTI, Michigan City, IN) and projected
on a video monitor. The images were then taken from the monitor with a
frame grabber card (model MVP AT, Matrox Electronic Systems, Dorval,
PQ). The software used for these measurements was Optimas, which was
obtained from Bioscan (Edmonds, WA). With this software, the computer
was allowed to define the areas of interest and to compute the total
(integrated) gray value, which is a measure of both the intensity and
the area of color. Individual iNOS mRNA signal levels were then divided
by the corresponding 28S rRNA sample signal level to normalize the
values for the amount of RNA loaded. For each experiment, the iNOS mRNA
level for cells exposed to LPS alone was taken to be 100%. The iNOS
mRNA level for cells exposed to both LPS and surfactant is expressed as
a percentage of that for cells exposed to LPS alone. In all cases, the
range of darkness for the scanned images was between 30 and 75% of the
full scale (100% = darkest). Therefore, image analysis was performed
in the midrange of the darkness scale.
Detection of iNOS protein. To
determine whether iNOS protein could be detected in alveolar
macrophages exposed to LPS alone, to LPS plus lung surfactant, or to
LPS plus SP-B, Western blot analysis was used. Alveolar macrophages
were incubated for 22 h in culture medium with LPS, LPS plus pulmonary
surfactant, or LPS plus SP-B as described in
Measurement of · NO
production. After the incubation period, the
supernatants were removed and saved for analysis of nitrate and
nitrite. The cells were removed from the culture plates and used for
Western blot analysis. SDS-PAGE was performed on 100-µg aliquots of
cell protein with 7.5% (wt/vol) polyacrylamide gels. Proteins were
transferred to nitrocellulose paper with an electrophoretic transfer
unit (Hoefer Scientific Instruments, San Francisco, CA). The blots were
then blocked for 1 h at room temperature in a medium (blocking buffer)
containing 50 mM Tris · HCl, 150 mM NaCl, 2%
(vol/vol) BSA, and 0.1% (vol/vol) Tween 20, pH 7.4. These blots were
then incubated for an additional hour at room temperature in blocking
buffer containing anti-iNOS antibody. The primary antibody used was
mouse macrophage (IgG2a) monoclonal anti-iNOS (Transduction
Laboratories, Lexington, KY). The antibody was diluted 1:500 in
blocking buffer. After incubation with the primary antibody, the blots
were washed six times (5 min/wash) at room temperature with medium
containing 50 mM Tris · HCl, 150 mM NaCl, and 0.1%
Tween 20 (Tris-buffered saline-Tween 20; pH 7.4). Then the blots were
incubated for 1 h at room temperature in blocking buffer containing the
secondary antibody, anti-mouse IgG coupled to horseradish peroxidase
(Amersham Life Sciences, Cleveland, OH). After incubation with the
secondary antibody, the blots were washed six times (5 min/wash) at
room temperature with Tris-buffered saline-Tween 20. Protein bands
detected by the antibody were visualized by enhanced chemiluminescence
(Amersham Life Sciences). The standard that was carried through the
entire procedure was macrophage lysate prepared from RAW 264.7 cells that had been stimulated with IFN-
and LPS (Transduction
Laboratories). To estimate the relative amounts of iNOS protein in the
alveolar macrophages, the Western blots were subjected to image
analysis as described in Detection of iNOS
mRNA. For each experiment, the protein level for the
cells exposed to LPS alone was taken to be 100%. The iNOS protein
level for cells exposed to LPS plus surfactant or to LPS plus SP-B is
expressed as a percentage of that for cells exposed to LPS alone.
Measurement of iNOS activity. The
effects of pulmonary surfactant on LPS-induced iNOS activity in
alveolar macrophages were studied by measuring the conversion of
L-arginine to
L-citrulline (6). Alveolar
macrophages were incubated with LPS for 22 h in culture medium as
described in Measurement of · NO
production. After the incubation period, the cells were
removed from the culture plates and suspended in ice-cold medium
containing 50 mM Tris · HCl, 12 mM mercaptoethanol,
0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, 2 µM leupeptin, 1 µM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride, pH 7.0. A
crude preparation of cellular cytosol and microsomes was made by
sonicating the suspension twice for 10 s each time and then spinning at
13,000 g for 1 min in an Eppendorf microfuge. The supernatants were removed and frozen at
80°C
until used for assay.
To measure conversion of
L-arginine to
L-citrulline, 50 µl of the
cell cytosolic and microsomal preparation (containing 50 µg of
protein) were added to 50 µl of a medium containing 67 mM Tris · HCl, 3.3 mM
CaCl2, 2.0 mM NADPH, 4 µM flavin
mononucleotide, 4 µM FAD, 20 U of calmodulin, and 20 µM tetrahydrobiopterin, pH 7.2. Some samples contained either
pulmonary surfactant (25 µg PL/ml) or aminoguanidine (1 mM). The
reaction was initiated by adding 0.15 µCi of
L-[2,3,4,5-3H]arginine
monohydrochloride (specific activity 61 Ci/mmol; Amersham Life
Sciences). Incubations were carried out at 37°C for 10 min. The
concentration of protein and time of incubation used were within linear
ranges. After the incubation period, 400 µl of sodium HEPES buffer
(30 mM sodium HEPES and 3 mM EDTA) were added to the incubation mixture
that was then placed on ice. Then 400 µl of a 50% (wt/vol) slurry
of Dowex (Dowex AG 50W-8X, sodium form, Bio-Rad Laboratories, Hercules,
CA) were added to the samples, and the samples were vortexed for 20 s.
The Dowex was removed by spinning the samples at 13,000 g for 2 min in the microfuge. An
aliquot (0.5 ml) of the supernatant was then added to 4 ml of
scintillation fluid (EnviroSafe, Anoroc Scientific, Hackensack, NJ),
and the samples were counted in a liquid scintillation counter. The
results are expressed as picomoles of
L-citrulline produced per
milligram of protein.
Generation of · NO by spermine/NO
complex. · NO was produced by a
· NO generator in a cell-free system to determine whether pulmonary surfactant can act as an antioxidant or radical scavenger and
reduce the amount of · NO formed. Spermine/NO complex (RBI) was incubated for 1 h in culture medium maintained at 37°C in 95%
air-5% CO2 (relative humidity
90%). The final concentration of spermine/NO complex was 0.2 mM
because this concentration produces approximately the same amount of
· NO as that formed by 2 × 106 LPS-stimulated alveolar
macrophages, the cell number used in 1 ml of all our incubation
mixtures. Incubations were carried out with spermine/NO complex alone
or with spermine/NO complex plus lung surfactant (200 µg PL/ml).
After the incubation period, the samples were removed, and nitrate and
nitrite was measured as described in Measurement of
· NO production.
Statistical analyses. All comparisons
of significance were made by comparing each treatment with the control
value (100%) with a one-sample Student's
t-test.
P < 0.05 was taken as the limit to
indicate significance.
 |
RESULTS |
NOS inhibitor effects on LPS-induced · NO
production. The effects of three different NOS
inhibitors on LPS-induced · NO formation by alveolar
macrophages were determined, and the results are shown in Fig.
1. Incubation of the cells with
L-NAME,
L-NMMA, or aminoguanidine led to
inhibition of LPS-induced · NO production by 71, 93, or 83%,
respectively. These results are much different from the inhibitor effects on the · NO produced by a constitutive NOS (cNOS) in
rat alveolar macrophages, where only
L-NAME inhibited production by only 32% (27). L-NAME and
L-NMMA are inhibitors of both
iNOS and cNOS (35), whereas aminoguanidine is specific for iNOS (13). Previous results by Miles et al. seem to confirm the
specificity of aminoguanidine for iNOS in lung cells in that it did not
inhibit · NO formed by cNOS in either alveolar macrophages
(27) or alveolar type II cells (26). Thus all of these results appear
to confirm that the · NO produced by alveolar macrophages in
response to LPS is due to iNOS.

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Fig. 1.
Effects of nitric oxide (· NO) synthase inhibitors on
lipopolysaccharide (LPS)-induced · NO production by alveolar
macrophages. Cells (2 × 106/ml) were incubated for 22 h in
culture medium (MEM supplemented with glutamine, HEPES,
penicillin-streptomycin, kanamycin, and fetal bovine serum) maintained
at 37°C in 95% air-5% CO2
(relative humidity 90%). Alveolar macrophages were incubated with LPS
(10 µg/ml) alone or with
LPS+NG-monomethyl-L-arginine
(L-NMMA),
NG-nitro-L-arginine
methyl ester (L-NAME), or
aminoguanidine (AMG). Final concentration of all inhibitors was 1 mM.
After incubation period, nitrate and nitrite in supernatants were
measured as described in METHODS.
Values are means ± SE for 6 experiments and are expressed as
percentage of · NO production by cells exposed to LPS alone
(%LPS induced). Nitrate and nitrite production by cells exposed to LPS
alone was 108 ± 4 nmol/106
cells.
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Pulmonary surfactant effects on LPS-induced
· NO production. The effects of pulmonary
surfactant on · NO production by alveolar macrophages were
investigated. When alveolar macrophages were incubated in culture
medium alone for 22 h, very little nitrate plus nitrite was formed,
i.e., 0.36 ± 0.12 (SE)
nmol/106 cells
(n = 6 experiments). If the cells were
incubated with lung surfactant (200 µg PL/ml), more nitrate plus
nitrite was produced, i.e., 3.9 ± 1.9 nmol/106 cells. However, when the
cells were incubated with LPS (10 µg/ml), there was a great increase
in the nitrate plus nitrite formed, i.e., 108 ± 4 nmol/106 cells. Incubation of
alveolar macrophages with LPS in the presence of lung surfactant led to
a concentration-dependent inhibition of LPS-induced · NO
formation. These results are shown in Fig. 2. · NO production was inhibited by ~50% at surfactant
levels of ~100 µg PL/ml and by ~75% at the highest surfactant
level used, 200 µg PL/ml. These results demonstrate that pulmonary
surfactant inhibits LPS-induced · NO production by alveolar
macrophages in a concentration-dependent manner. Many of the remaining
experiments described in this paper were designed to study the
mechanism by which surfactant inhibits · NO formation. In
these experiments, the concentration of surfactant used was 200 µg
PL/ml.

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Fig. 2.
Effects of different amounts of pulmonary surfactant on LPS-induced
· NO production by alveolar macrophages. Cells (2 × 106/ml) were incubated for 22 h in
culture medium (MEM supplemented with glutamine, HEPES,
penicillin-streptomycin, kanamycin, and fetal bovine serum) maintained
at 37°C in 95% air-5% CO2
(relative humidity 90%). Some cells were exposed to LPS (10 µg/ml)
alone, and other cells were exposed to LPS (10 µg/ml) and varying
amounts of lung surfactant. Amounts of lung surfactant are expressed as
µg phospholipid (PL)/ml. After incubation period, nitrate and nitrite
in supernatants were measured as described in
METHODS. Values are means ± SE for
6 experiments and are expressed as percentage of that produced in
presence of LPS alone (%control). Nitrate and nitrite production by
cells exposed to LPS alone was 108 ± 4 nmol/106 cells.
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Experiments were performed to determine whether surfactant inhibits
LPS-induced · NO production by interfering with the ability of LPS to initiate the events involved in the induction of iNOS, e.g.,
the binding of LPS to the cell membrane. In these experiments, we
compared the ability of surfactant to inhibit · NO formation when the cells were incubated for 22 h with LPS and surfactant together
with its ability to inhibit · NO production when the cells
were exposed to surfactant after stimulation with LPS. In the latter
situation, the cells were incubated with LPS for 2 h. The LPS was then
washed from the cells, either medium alone or surfactant was added, and
the incubations were carried out for an additional 20 h. The results
are shown in Table 1. Although there is
less · NO produced by cells exposed to LPS for only 2 h, the
effects of surfactant are the same in both cases; i.e., surfactant
inhibits LPS-induced · NO production by 70-76%.
Therefore, these results show that surfactant does not inhibit
LPS-induced · NO production by interfering with the initial
events in the process, such as LPS binding to the membrane.
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Table 1.
Effects of altering incubation time with LPS and pulmonary surfactant
on · NO production by alveolar macrophages
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Pulmonary surfactant effects on iNOS
mRNA. Northern blot analysis was used to determine
whether lung surfactant interferes with LPS-induced formation of iNOS
mRNA. A representative Northern blot is shown in Fig.
3. This result demonstrates that there was no iNOS message in cells incubated in culture medium alone (Fig. 3,
lane 1). Exposure of the cells to
LPS led to a substantial level of iNOS mRNA (Fig. 3,
lane 2), and the addition of lung surfactant had no effect on LPS-induced iNOS mRNA (Fig. 3,
lane 3). All blots were subjected to
image analysis to estimate the relative amounts of iNOS mRNA in cells
exposed to LPS alone and in cells exposed to LPS plus surfactant. The
results are shown in Table 2. Incubation of
the alveolar macrophages with LPS plus surfactant led to iNOS mRNA
levels that were no different from the mRNA levels in the presence of
LPS alone. In addition, we also obtained similar results by performing
PCR on serial dilutions of reverse-transcribed DNA. Thus these results
show that lung surfactant had no effect on the amount of LPS-induced
iNOS mRNA in alveolar macrophages.

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Fig. 3.
Northern blot analysis of inducible · NO synthase (iNOS) mRNA
and 28S rRNA obtained from untreated alveolar macrophages
(lane 1) and cells treated with LPS
alone (lane 2) or LPS+pulmonary
surfactant (lane 3). Cells (2 × 106/ml) were incubated for
22 h in culture medium (MEM supplemented with glutamine, HEPES,
penicillin-streptomycin, kanamycin, and fetal bovine serum) maintained
at 37°C in 95% air-5% CO2
(relative humidity 90%). Alveolar macrophages were incubated alone
(control), with LPS (10 µg/ml), or with LPS+lung surfactant (200 µg
PL/ml). After incubation period, cells were removed from culture
plates, and total cellular RNA was isolated and analyzed for iNOS mRNA
expression by Northern blot analysis as described in
METHODS. Blot is representative of
results obtained from 7 different alveolar macrophage preparations.
|
|
Pulmonary surfactant effects on iNOS
protein. Experiments were performed in an attempt to
determine whether exposure of alveolar macrophages to pulmonary
surfactant led to changes in the amount of iNOS protein produced in
response to LPS. Western blot analysis was used. The results, which are
shown in Fig. 4, demonstrate the presence
of an LPS-induced alveolar macrophage protein that reacted with a
monoclonal anti-iNOS antibody (lane
3). The molecular mass of this protein
(~120 kDa) corresponds to that of an iNOS standard that was obtained
from macrophage lysate prepared from RAW 264.7 cells that had been
stimulated with IFN-
and LPS (Fig. 4, lane
2). No iNOS protein was detected in cells incubated
without LPS or with lung surfactant alone (data not shown). However,
when the cells were incubated with LPS plus surfactant, there was less iNOS protein present (Fig. 4, lane
4) than when the cells were incubated with LPS alone
(Fig. 4, lane 3). The blots were
subjected to image analysis to estimate the relative amounts of iNOS
protein present (Table 3). The results show
that incubation of alveolar macrophages with lung surfactant led to
80-90% decreases in LPS-induced iNOS protein levels and
· NO production. Therefore, the surfactant-induced inhibition
of LPS-stimulated · NO formation appears to be related to a
reduction in the amount of iNOS protein.

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Fig. 4.
Western blot analysis of alveolar macrophage proteins with anti-iNOS
antibody. Cells (2 × 106/ml)
were incubated for 22 h in culture medium (MEM supplemented with
glutamine, HEPES, penicillin-streptomycin, kanamycin, and fetal bovine
serum) maintained at 37°C in 95% air-5%
CO2 (relative humidity 90%).
Alveolar macrophages were incubated with LPS (10 µg/ml) alone or with
LPS+lung surfactant (200 µg PL/ml). After incubation period, cells
were removed from culture plates, and membranes were disrupted as
described in METHODS. SDS-PAGE was
used to fractionate 100-µg aliquots of cell protein. Proteins were
then transferred to a nitrocellulose membrane and immunodetected with a
monoclonal anti-iNOS antibody. Lane 1,
molecular-mass markers (nos. at
left); lane
2, standard preparation of iNOS from RAW 264.7 cells
that had been stimulated with LPS and interferon- was used as a
positive control and was carried through entire procedure;
lane 3, proteins from cells exposed to
LPS alone; lane 4, proteins from cells
exposed to LPS+lung surfactant. Arrow at right location of iNOS
at a molecular mass of ~120 kDa. Blot is representative of results
obtained from 6 different alveolar macrophage preparations.
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|
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Table 3.
Effects of pulmonary surfactant on LPS-induced · NO
production and iNOS protein levels in alveolar macrophages
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|
Pulmonary surfactant effects on iNOS
activity. To determine whether lung surfactant can
inhibit iNOS activity, the conversion of
L-arginine to
L-citrulline was measured in a
crude preparation of cytosol and microsomes obtained from alveolar
macrophages that had been incubated with LPS for 22 h. The results are
shown in Table 4. When the preparation was
incubated alone (with no surfactant), there were ~3 pmol
L-citrulline
produced/mg protein in 10 min. There was no change in iNOS activity
when lung surfactant was included in the incubation mixture. It should
be noted that we used the same ratio of lung surfactant to cellular
protein in these experiments as was used in the incubation of intact
alveolar macrophages with surfactant in the experiments described in
Measurement of iNOS activity. The iNOS
inhibitor aminoguanidine was used as a positive control. Aminoguanidine
(1 mM) led to a 98% reduction in iNOS activity. These results
demonstrate that lung surfactant does not affect iNOS activity in
LPS-induced alveolar macrophages, at least in vitro.
Pulmonary surfactant effects on · NO
generation by spermine/NO complex. · NO was
produced by an · NO generator in a cell-free system to
determine whether pulmonary surfactant can reduce the amount of
· NO formed by acting as an antioxidant or radical scavenger. Spermine/NO complex was used to generate · NO. The
concentration of the generator was adjusted so that the · NO
produced was approximately the same as that formed by the number of
LPS-stimulated cells placed in one well of the culture plates, i.e., as
in all other experiments in which · NO production was
measured. The amount of lung surfactant used in these experiments was
approximately the same as the greatest amount used to inhibit
· NO formation by the intact cells in one well of the culture
plates. The results are shown in Table 5.
· NO production by spermine/NO complex was not affected by
lung surfactant. Similar results were obtained with another
· NO generator,
S-nitroso-N-acetylpenicillamine
(data not presented). These results suggest that surfactant does
not inhibit · NO production from LPS-stimulated alveolar
macrophages by acting as an antioxidant or radical scavenger.
Effects of pulmonary surfactant components on
LPS-induced · NO production. Experiments were
performed in an attempt to determine which component(s) of lung
surfactant is responsible for the inhibition of LPS-induced
· NO production by alveolar macrophages. The results are
shown in Table 6. The effects of whole lung
surfactant, which produces ~75% inhibition at a concentration of 200 µg/ml, have been described (Fig. 2) and are shown here for the sake
of comparison. Incubation of the cells with the major component of
surfactant, DPPC, had no effect on LPS-induced · NO
formation. The concentration of DPPC used was 100 µg/ml because it
accounts for ~50% of the PLs in surfactant. We also determined that
incubating the cells with a mixture of some surfactant lipids (mixed
lipids; DPPC, unsaturated PC, PG, and cholesterol in a molar ratio of
10:5:2:3; final concentration 200 µg PL/ml) had no effect. The major
hydrophilic surfactant-associated protein, SP-A, also had no effect on
LPS-induced · NO production at concentrations up to 40 µg/ml. In addition, inclusion of both mixed lipids and SP-A in the
incubation medium had no effect. On the other hand, Survanta, a
commercial surfactant preparation used for replacement therapy,
inhibited LPS-induced · NO formation by ~40% at a
concentration of 200 µg PL/ml, i.e., the same PL concentration as was
used for whole lung surfactant.
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Table 6.
Effects of pulmonary surfactant components and Survanta on LPS-induced
· NO production by alveolar macrophages
|
|
Survanta contains some of the same lipid components that are present in
natural surfactant, and it also contains the hydrophobic SPs SP-B and
SP-C. There is no SP-A in Survanta. Thus it may be that the hydrophobic
proteins are responsible for the surfactant-induced inhibition of
· NO formation. Therefore, experiments were performed to
determine the effects of SP-B and SP-C on LPS-induced · NO production by alveolar macrophages. For these experiments, different amounts of the proteins were incorporated into mixed lipid vesicles (200 µg PL/ml) for delivery to the cells. Incubation of alveolar macrophages with SP-C at concentrations up to 20 µg/ml had no effect
on LPS-induced · NO production; i.e., · NO
formation in the presence of LPS plus SP-C was 99 ± 4% of that in
the presence of LPS alone (n = 6 experiments). On the other hand, LPS-induced · NO formation
is inhibited by SP-B in a concentration-dependent manner (Fig.
5). · NO production is inhibited
by ~50 and ~90% at SP-B concentrations of 1-2 and 10 µg/ml,
respectively. Therefore, all of these results taken together indicate
that the component responsible for surfactant-induced inhibition of
LPS-stimulated · NO production is SP-B.

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Fig. 5.
Effects of different concentrations of surfactant protein (SP) B on
LPS-induced · NO production by alveolar macrophages. Cells (2 × 106/ml) were incubated for
22 h in culture medium (MEM supplemented with glutamine, HEPES,
penicillin-streptomycin, kanamycin, and fetal bovine serum) maintained
at 37°C in 95% air-5% CO2
(relative humidity 90%). Some cells were exposed to LPS (10 µg/ml)
alone, and other cells were exposed to LPS (10 µg/ml) and varying
amounts of SP-B. SP-B was incorporated into lipid vesicles for delivery
to cells as described in METHODS.
Lipid vesicles alone had no effect on · NO production. After
incubation period, nitrate and nitrite in supernatants were measured as
described in METHODS. Values are means ± SE for 6 experiments and are expressed as percentage of that
produced in presence of LPS alone. Nitrate and nitrite production by
cells exposed to LPS alone was 98 ± 6 nmol/106 cells.
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|
SP-B effects on iNOS protein.
Incubation of alveolar macrophages with lung surfactant led to
inhibition of LPS-stimulated · NO formation and iNOS protein
levels. The data presented in Effects of pulmonary
surfactant components on LPS-induced · NO production show that SP-B inhibited LPS-induced
· NO formation. Therefore, experiments were performed to
determine the effects of SP-B on the amount of LPS-induced iNOS protein
in alveolar macrophages. Western blot analysis was used. The results,
which are shown in Table 7, demonstrate
that incubation of the cells with SP-B (10 µg/ml, an amount that
produces ~90% inhibition of LPS-induced · NO production)
led to an ~80% reduction in LPS-induced iNOS protein levels. Thus
this result suggests that the surfactant component responsible for
surfactant-induced reduction in LPS-induced iNOS protein is SP-B.
 |
DISCUSSION |
The results of our experiments demonstrate that pulmonary surfactant
inhibits LPS-induced · NO production by rat alveolar macrophages in a concentration-dependent manner. At the highest level
of surfactant used (200 µg PL/ml), LPS-induced · NO
formation is reduced by 75-85%. The inhibition appears to be due
to a surfactant-induced reduction in the amount of enzyme responsible
for · NO production, i.e., iNOS. In the presence of
surfactant, iNOS protein levels are reduced by ~80%. The component
of surfactant that appears to be responsible for inhibiting LPS-induced
· NO formation is SP-B, one of the surfactant-associated
hydrophobic proteins. SP-B inhibits · NO production in a
concentration-dependent manner. At the highest SP-B level we used, 10 µg/ml, · NO formation was reduced by ~90%.
In addition to a reduction in iNOS protein levels, there are some other
possible ways by which surfactant could inhibit LPS-induced · NO formation. However, our results seem to rule out these
possibilities. Experiments in which alveolar macrophages are stimulated
with LPS before the addition of surfactant suggest that surfactant does
not interfere with the initial events in the process of iNOS induction
by LPS, such as LPS binding to the membrane (Table 1). This is also
supported by the fact that iNOS mRNA levels are not reduced in the
presence of surfactant (Table 2). We have also obtained evidence that
surfactant does not directly affect iNOS activity by showing that it
has no effect in vitro on the enzyme obtained from LPS-stimulated cells
(Table 4). Finally, surfactant has no effect on the · NO
produced by a cell-free generating system, spermine/NO complex (Table
5). This result indicates that the surfactant reduction in
· NO formation is probably not due to an antioxidant or
radical scavenging effect. Thus all of these data taken together
suggest that surfactant inhibits LPS-induced · NO production
by causing a decrease in alveolar macrophage iNOS protein levels.
Our data suggest that the effects of surfactant on iNOS protein levels
may be posttranscriptional and/or posttranslational. We have
shown that iNOS mRNA levels are not affected by surfactant. It may be
that the message is not translated so that iNOS protein synthesis does
not occur. If this is the case, the surfactant effect would be
posttranscriptional. Under normal conditions, the iNOS protein level is
determined by a balance between its rate of synthesis and its rate of
degradation. Thus another possibility is that translation occurs so
that iNOS protein is synthesized and that its reduction is due to an
increase in the rate of degradation. In this case, the effect would be
posttranslational. Of course, it is also possible that the surfactant
effect is a combination of posttranscriptional and posttranslational
effects. At this time, it is not possible to ascertain which of these
effects is responsible for surfactant-induced inhibition of
· NO production.
The surfactant component responsible for the inhibition of LPS-induced
· NO production appears to be SP-B. This protein inhibits · NO formation in a concentration-dependent manner. At the
highest concentration we used, 10 µg/ml, · NO production
was inhibited by ~90%. Hull et al. (16) measured the concentrations
of SP-B and PL in human bronchoalveolar lavage fluid. Their results
show that the weight ratio of SP-B to PL is ~1:10. If this is the
case for our surfactant preparations, an SP-B concentration of 10 µg/ml is approximately one-half of that present in the highest
surfactant levels used in our incubations (i.e., 200 µg PL/ml). These
calculations suggest that there is sufficient SP-B in surfactant to
account for the inhibition of LPS-induced · NO formation.
Furthermore, we calculated that the SP-B level in natural surfactant is
3- to 10-fold greater than that in Survanta (Miles and Baatz, personal observations), which probably accounts for the greater inhibitory effect of natural surfactant. None of the other surfactant
components we studied has any effect on · NO production. The
major lipid components, SP-A, and SP-C had no effect. The other
hydrophilic surfactant protein, SP-D, was not examined in the present study.
Almost all previous studies done with regard to SP-B have been
concerned with its role in surfactant metabolism and function (see Ref.
15 for a review). For example, it has been shown that this protein is
important in the intra-alveolar ordering of surfactant lipids into
tubular myelin after their secretion from alveolar type II cells (34,
37). SP-B also plays an important role in making the surfactant readily
spreadable and in stabilizing the film at the air-liquid interface (10,
38). Also, this protein stimulates the reuptake of phospholipids by
type II cells (9, 36), suggesting that it is involved in surfactant
reutilization. As far as we know, the results of our present study with
regard to SP-B are unique in that they represent the first reported
effect of SP-B that apparently is not directly related to surfactant metabolism or function. It also represents the first reported effect of
this protein on alveolar macrophages and suggests that SP-B may play a
role in lung defense.
The exact mechanism by which SP-B inhibits LPS-induced · NO
production is not known. We have shown that there is a reduction in the
iNOS protein level and suggested that this effect may be posttranscriptional and/or posttranslational. It may be that
SP-B is taken into the cells along with surfactant lipids and then exerts its effects intracellularly. In this regard, Miles et al. (29)
have shown that natural surfactant and lipid vesicles are readily taken
up to the same extent by alveolar macrophages. Furthermore, Breslin and
Weaver (7) studied the uptake of SP-B by alveolar type II cells. Their
data suggest that SP-B is internalized by a pathway similar to PLs.
These results suggest that the protein may exert its effect inside the
cells. Alternatively, SP-B may exert its effects via a cell surface
receptor, although an SP-B-specific macrophage cell surface receptor
has not been identified as yet.
One result from our study differs from that reported by Blau et al.
(5). They found that · NO production by rat alveolar macrophages is upregulated by SP-A. One source of their SP-A was human
alveolar proteinosis patients. In this regard, our SP-A was obtained
from a similar source and isolated in a manner similar to that used by
Blau et al. However, SP-A had no effect on · NO formation by
alveolar macrophages in our hands. In most of our experiments, we used
an SP-A concentration of 20-40 µg/ml, whereas Blau et al. found
a maximal effect at 4 µg/ml. When we did use SP-A concentrations as
low as 4 µg/ml, there was no effect on cellular · NO
production (data not shown). The reasons for these contrasting effects
of SP-A are not known.
The significance of surfactant effects on LPS-induced · NO
formation by alveolar macrophages is not known. However, other investigators have reported that various surfactant preparations or
components of surfactant have inhibitory effects on the release of
cytokines from alveolar macrophages. For example, surfactant preparations used in replacement therapy, Exosurf and Survanta, suppress the endotoxin-induced production of three cytokines, TNF-
,
IL-1
, and IL-6 (39, 40). The major hydrophilic SP, SP-A, also
inhibits the release of TNF-
from LPS-stimulated alveolar macrophages (25). It is known that TNF-
is involved in initiating the inflammatory cascade (3) and that the release of · NO due to induction of iNOS is part of the inflammatory process. Furthermore, surfactant inhibits the production of TNF-
and · NO by
LPS-stimulated cells. Because surfactant is part of the normal
environment for alveolar macrophages in vivo, it is possible that it
serves to attenuate some of the inflammatory processes. The local
concentration of surfactant around the cells on the alveolar surface is
not known, so it is not possible to determine the amount of attenuation that occurs in vivo. All of these findings taken together suggest that
surfactant may play a protective role in the lungs by reducing the
amount of inflammation and restricting lung damage. This latter idea
was suggested by McIntosh et al. (25) with regard to SP-A effects in
alveolar macrophages.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: P. R. Miles, NIOSH, HELD/MS/2015,
1095 Willowdale Rd., Morgantown, WV 26505.
Received 7 August 1998; accepted in final form 8 October 1998.
 |
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