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Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan;
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan; and
Lord and Tayler Laboratory for Lung Biochemistry, Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induced
by smooth LPS, but rough LPS-induced TNF- expression was unaffected
by SP-A. When U937 cells and rat alveolar macrophages were preincubated
with SP-A, smooth LPS failed to induce TNF- secretion, whereas rough
LPS-induced TNF- secretion was modestly increased. To clarify the
mechanism by which SP-A modulates LPS-elicited cellular responses, we
further examined the interaction of SP-A with CD14, which is known as a
major LPS receptor. Western blot analysis revealed that CD14 was one of
the SP-A binding proteins isolated from solubilized U937 cells. In
addition, SP-A directly bound to recombinant soluble CD14 (rsCD14).
When rsCD14 was preincubated with SP-A, the binding of rsCD14 to smooth
LPS was significantly reduced but the association of rsCD14 with rough
LPS was augmented. These results demonstrate the different actions of
SP-A upon distinct serotypes of LPS and indicate that the direct
interaction of SP-A with CD14 constitutes a likely mechanism by which
SP-A modulates LPS-elicited cellular responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS is a major constituent of the outer membrane of Gram-negative bacteria. Most enteric bacteria express smooth LPS, which is composed of O-Ag, complete core oligosaccharides, and the endotoxin-principal region, lipid A. There are also rough mutants such as Ra, Rb, Rc, Rd, and Re strains which lack O-Ag but possess lipid A and progressively shorter core oligosaccharides (16). A number of Gram-negative bacteria colonizing the surfaces of the respiratory tract express a rough LPS phenotype (17). Direct interaction between SP-A and rough LPS has been suggested (18), but its physiological and functional significance has not been clearly defined.
LPS has been known to activate macrophages and to induce a
variety of mediators including TNF-, IL-1, IL-6, IL-8, IL-12,
migration inhibitory factor, chemokine, IFN, eicosanoids, and reactive
oxygen (19, 20). The response to physiological amounts of LPS by
reactive cells depends on membrane CD14 (mCD14) (20), which is a 55-kDa
glycoprotein that is GPI-anchored to the plasma membrane of myeloid
cells. A soluble form of the protein (sCD14) is also found in plasma
and facilitates the responsiveness of cells to LPS (21, 22). However,
the mechanism by which the LPS signal is transduced across the cell
membrane remains unknown. It has been considered that the role of mCD14
is to bind LPS and the subsequent interaction with an unidentified
transmembrane protein is required for transmitting LPS signal (23).
Recently, it has been suggested that Toll-like receptor 2 is a direct
mediator of signaling by LPS (24).
It is physiologically quite important to understand the role of surfactant in host defense against respiratory infections. The lungs are continually exposed to ambient air that contains significant numbers of microorganisms. Because surfactant covers all of the alveolar surfaces, any inhaled pathogens must interact with surfactant before interactions with target cells. Therefore, we focused on the interactions of SP-A with LPS and the consequences of the interaction upon the LPS-elicited responses in macrophage-like cell line U937 cells and alveolar macrophages. Our findings demonstrate that SP-A and the region of the neck plus CRD bind rough but not smooth LPS. The cellular response to smooth LPS is prevented by SP-A, but the response to rough LPS can be enhanced. We propose that SP-A modulates LPS-induced cellular responses via its direct interaction with CD14.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Smooth LPS (Escherichia coli O26:B6, O111:B4), rough LPS (Salmonella minnesota Re595, E. coli J5), and polymyxin B-agarose were purchased from Sigma (St. Louis, MO). Lipid A from S. minnesota Re595 was obtained from List Biologic Laboratories (Campbell, CA). N-hydroxysulfosuccinimidobiotin (Sulfo-NHS-biotin) was from Pierce (Rockford, IL), CNBr-activated Sepharose 4B was from Pharmacia Biotech (Uppsala, Sweden), and chemiluminescence reagents was purchased from NEN (Boston, MA). mAb to human CD14 (12C3) was obtained from Boehringer Mannheim (Mannheim, Germany). Macrophage-like cell line U937 cells (JCRB9021) were obtained from the Health Science Research Resources Bank (Osaka, Japan). L929 cells were kindly provided by Dr. Kazuko Kajiyama (Chugai Pharmaceutical, Tokyo, Japan). The cells were maintained in RPMI 1640 medium (Nissui, Tokyo, Japan) containing 10% FCS.
SP-A
Surfactant was isolated from bronchoalveolar lavage fluids of patients with alveolar proteinosis as described previously (25). After the surfactant was delipidated with 1-butanol (26), SP-A was purified from the delipidated surfactant by mannose-Sepharose 6B column chromatography followed by gel filtration over a Bio Gel A5m column (BioRad Laboratories, Richmond, CA) (27). The collagenase-resistant fragment (CRF) of human SP-A was prepared as described previously (28).
Monitoring and removal of endotoxin in SP-A preparations
Endotoxin in SP-A preparations was removed by polymyxin B-agarose in the presence of octyl-ß-D-glucoside as described by McIntosh et al. (15). Endotoxin levels in untreated or polymyxin-treated SP-A preparations were 55 pg or below 0.5 pg/µg of protein, respectively, when measured by chromogenic assay using Limulus amebocyte lysate system (ENDOSPECY; Seikagaku Kogyo, Tokyo, Japan). Polymyxin-treated SP-A was used for the experiments performed in this study.
Binding of SP-A to LPS
LPS (5 µg/well) or lipid A in 20 µl of ethanol was placed in microtiter wells (Immulon 1B; Dynex Laboratories, Chantilly, VA), and the solvent evaporated in ambient air. After the nonspecific binding was blocked with 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2, and 1 mg/ml of BSA (buffer A), various concentrations of human SP-A (hSP-A) or CRF in 50 µl of the buffer A were added and incubated at 37°C for 5 h. The wells were then washed with buffer A, and 20 µg/ml anti-human SP-A IgG (50 µl/well) in PBS containing 0.1% (v/v) Triton X-100 and 3% (w/v) skim milk (buffer B) was added and incubated for 1 h, followed by the incubation with HRP-labeled anti-rabbit IgG (1:1000) for 1 h. After washing the wells with PBS containing 0.1% (v/v) Triton X-100, the peroxidase reaction was finally performed using o-phenylenediamine as a substrate. The binding of SP-A to LPS was detected by measuring absorbance at 492 nm.
Aggregation of LPS
LPS aggregation was conducted by a modified method based on that described by van Iwaarden et al. (18). LPS or lipid A (25 µg/ml) in 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl was probe-sonicated and incubated with or without hSP-A (20 µg/ml) for 5 min at room temperature. After the initial absorbance reading at 400 nm by a Hitachi U-2000 spectrophotometer (Tokyo, Japan), CaCl2 was added to a final concentration of 1 mM at a time of 3 min and the absorbance was further measured until a time of 10 min.
TNF- mRNA expression
U937 cells (0.5 x 106) were induced
to differentiate by incubation with 10 nM PMA for 24 h. The cells
were further incubated in the absence of PMA for 24 h in RPMI 1640
medium containing 10% FCS. The indicated amounts of LPS were added to
the cell culture and incubated in the absence or presence of SP-A for
2 h at 37°C with 5% CO2. After
incubation, total cellular RNA was isolated from the cells by acid
guanidinium thiocyanate-phenol-chloroform method (29). A cDNA pool was
obtained from 1 µg of RNA using 200 U Superscript II Reverse
Transcriptase (Life Technologies, Gaithersburg, MD) and 0.5 µg
oligo(dT)12–18 primer (Life Technologies). The
region of cDNA for human TNF- was amplified from the cDNA pool
derived from U937 cells by PCR using 2.5 nM specific primers
(5'-AAGCCTGTAGCCCATGTTGT-3' as a sense primer and
5'-CAGATAGATGGGCTCATACC-3' as an antisense primer). PCR was
performed in the presence of 2 mM MgCl2, 0.2 mM
dNTPs, 1.25 U Taq polymerase, and 1 µCi of
[32P]dCTP. After the initial incubation at
95°C for 5 min, 25 cycles of amplification were conducted with
denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and
extension at 72°C for 2 min. PCR products (330 bp) were
electrophoresed on a 1.0% agarose gel, dried, and exposed to the
imaging plate for 1 h, and then radioactivities of PCR products
were measured with a Bio-Imaging Analyzer BAS 2000 (Fuji Photo Film,
Tokyo, Japan). TNF- mRNA was expressed as a relative ratio compared
with the radioactivity of a control PCR product derived from the cells
that had been incubated in the absence of LPS and SP-A.
TNF- secretion
TNF- secretion into medium was measured using an L929 cell
bioassay performed by a modified method based on that described by
Flick et al. (30). The L929 cells were seeded into 96- well plates
(6 x 104/well) in 100 µl/well of RPMI
1640 containing 10% FCS and 2 µg/ml actinomycin D (Sigma). Dilutions
of standard rTNF- (1–50 pg/ml) (PeproTech, Rocky Hill, NJ) or
samples (1:10 and 1:50) in a volume of 100 µl/well were added and the
cells were incubated at room temperature for 15 min followed by
incubation for overnight at 37°C with 5% CO2.
The next day the medium was removed and the cells were stained with
0.2% (w/v) crystal violet for 10 min. The wells were then washed with
water, and 100 µl/well of 33% acetic acid was added to extract the
retained crystal violet. The absorbance at 570 nm was finally
measured.
Effect of SP-A upon LPS-induced TNF- expression in U937 cells
Differentiated U937 cells (0.5 x
106) were incubated with 1–1000 ng/ml of smooth
or rough LPS in the presence of 0–50 µg/ml of hSP-A for the
indicated time. After the incubation, the medium was collected and
TNF- secretion was measured using an L929 cell bioassay as described
above. TNF- mRNA expression in U937 cells was also evaluated by
RT-PCR as described above. In some experiments, SP-A was preincubated
with the cells before the addition of LPS and then TNF- secretion
was measured.
Binding of SP-A to U937 cells
The binding study was adapted from that described for type II cell binding (27). Rat SP-A was iodinated by the method of Bolton and Hunter (31) using Bolton-Hunter reagent (Amersham, Arlington Heights, IL). The specific radioactivity ranged from 490 to 518 cpm/ng, and >84% of the radioactivity was precipitated by treatment with 10% (w/v) TCA. For the binding assay, differentiated U937 cells (0.5 x 106) in RPMI 1640 containing 10% FCS were incubated with various concentrations of the labeled protein at 37°C for 5 h. The cell monolayers were then washed with ice-cold 20 mM Tris buffer (pH 7.4) containing 0.1 M NaCl, 2 mM CaCl2, and 1 mg/ml BSA and were finally dissolved in 2 ml of 0.1 M NaOH. The amount of the labeled protein was determined using a gamma-radiation counter.
Biotinylation and solubilization of U937 cells
Differentiated U937 cells (150 x 106) were harvested, washed with ice-cold PBS (pH 7.4) by centrifugation at 1000 rpm, and incubated for 30 min at room temperature in 0.5 ml PBS containing N-hydroxysulfosuccinimido biotin (Sulfo-NHS-biotin) (Pierce) at a final concentration of 0.5 mg/ml. The cells were then washed with PBS to remove excess biotin, and the final cell pellet was solubilized by the incubation on ice for 30 min with 2 ml of 50 mM HEPES buffer (pH 7.4) containing 0.15 M NaCl, 1% Nonidet P-40, 1 mM PMSF, 1 mM EDTA, 1 µg/ml pepstatin A, 100 µg/ml benzamidine, 2 µM phosphoramidon, 10 µg/ml trypsin inhibitor, and 1 µg/ml aprotinin. Whole cell extracts were then centrifuged for 10 min at 400 x g, and the supernatant was further centrifuged for 30 min at 100,000 x g to separate detergent-insoluble fraction from detergent-soluble membranes.
Isolation and Western blot analysis of SP-A binding proteins on U937 cells
To prepare a hSP-A affinity column, 2.5 mg of hSP-A in 0.1 M NaHCO3 buffer (pH 8.3) containing 0.5 M NaCl was coupled with 0.5 g of CNBr-activated Sepharose 4B gel according to the manufacturer’s instruction (Pharmacia Biotech). The fraction solubilized from biotinylated cell membranes described above, which had been diluted with 10x volume of binding buffer (20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2, and 0.1% Nonidet P-40), was applied to hSP-A affinity column at 4°C. After the column was washed with the binding buffer, the bound materials were eluted with 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl, 10 mM EDTA, and 0.1% Nonidet P-40. The eluate was then concentrated and dialyzed against 5 mM Tris buffer (pH 7.4). All the procedures described above were performed under sterile conditions. To visualize the SP-A binding proteins isolated from U937 cells, the eluted samples were electrophoresed under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking PVDF membranes with the buffer B, the membranes were incubated with HRP-conjugated streptavidin D (1:1000) for 20 min. The membranes were washed, followed by the peroxidase reaction using diaminobenzidine tetrahydrochloride as a substrate. For the Western blot analysis, the PVDF membranes on which the SP-A binding proteins were transferred were incubated with either 20 µg/ml of mAb to CD14 or control mouse serum (1:500) for 90 min. The membranes were then washed with blocking buffer, followed by the incubation with HRP-labeled anti-mouse IgM (1:500) for 1 h. After the incubation, the membranes were washed with PBS containing 0.1% (v/v) Triton X-100. They were then incubated with chemiluminescence reagent (NEN) and the proteins were visualized on an x-ray film.
Binding of SP-A to rsCD14
Expression and purification of recombinant human soluble CD14 (rsCD14) from the culture supernatant of Pichia pastoris will be described elsewhere (S. Nomura and T. Muta, unpublished data). For the ligand blot analysis, 5 µg/lane of rsCD14 was electrophoresed under reducing conditions and transferred to PVDF membrane. After nonspecific binding was blocked with buffer B, the membrane was incubated with either 20 µg/ml of hSP-A or BSA in buffer B at room temperature overnight. The membranes were then washed with buffer B and incubated with anti-SP-A IgG (20 µg/ml) for 90 min, followed by the incubation with HRP-labeled anti-rabbit IgG (1:1000) for 75 min. SP-A that had bound to the PVDF membrane was visualized by using chemiluminescence reagent (NEN) as described above. rsCD14 transferred to the membrane was also visualized by Coomassie brilliant blue staining.
To examine the concentration-dependent binding of SP-A to CD14, 50 µl/well of sterile BSA or rsCD14 at a concentration of 10 µg/ml in 5 mM Tris buffer (pH 7.4) was coated onto microtiter wells and incubated at room temperature for 5 h. Nonspecific binding was blocked with 10 mM HEPES buffer (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2, and 5% (w/v) BSA (buffer C) and the indicated concentrations of hSP-A in 50 µl of buffer C were added and incubated at 37°C overnight. The wells were then washed with buffer B. Anti-SP-A IgG (20 µg/ml) was added (50 µl/well) and incubated at 37°C for 90 min, followed by the incubation with HRP-labeled anti-rabbit IgG (1:1000) for 75 min. The wells were washed with PBS containing 0.1% (w/v) Triton X-100 and the peroxidase reaction was performed using o-phenylenediamine as a substrate. The binding of hSP-A to sCD14 was detected by measuring absorbance at 492 nm.
Binding of rsCD14 to LPS
Two micrograms per well of smooth or rough LPS in 20 µl of ethanol was added into microtiter wells and air-dried. Nonspecific binding was blocked with buffer C. rsCD14 (5 µg/ml) in buffer C containing 2% (v/v) human serum in the absence or the presence of hSP-A (50 µg/ml) was preincubated at 37°C for 1 h. The preincubated protein mixtures (50 µl/well) were then added into the wells and further incubated with solid phase LPS at 37°C for 6 h. The binding of rsCD14 to LPS was detected by using polyclonal antisera (1:400) raised against recombinant CD14 expressed in Escherichia coli as a fusion protein with GST and HRP-labeled anti-rabbit IgG (1:1000).
Effect of SP-A upon LPS-induced TNF- secretion in alveolar
macrophages
Rat alveolar macrophages were obtained from bronchoalveolar
lavage fluids of Sprague Dawley rats. The lungs were lavaged with PBS
containing 0.5 mM EDTA. Isolated macrophages (1.0 x
105/well in a 96-well plate) were incubated
overnight in RPMI 1640 medium containing 10% FCS. Then, macrophages
were incubated in RPMI medium containing 10% FCS in the presence or
the absence of 10 µg/ml of hSP-A for 1 h, and then further
incubated with 2 ng/ml of LPS for 5 h. TNF- secretion into
medium was measured using a L929 cell bioassay as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To clarify the interaction of SP-A with endotoxin, we initially
examined the direct binding of SP-A to distinct structural types of LPS
coated onto microtiter wells (Fig. 1
A). hSP-A bound to lipid A
and rough strains of LPS. The binding of hSP-A to Rc strain (E.
coli J5) of rough LPS was significant but weaker than that to Re
strain (S. minnesota Re595). In contrast, hSP-A did not show
any significant binding to either of two strains of smooth LPS
(E. coli O26:B6 or O111:B4). The CRF of hSP-A, which
consists of the neck plus the CRD of SP-A, also bound to lipid A and Re
LPS although its binding appeared weaker than that of hSP-A (Fig. 1B). In this assay, when 5 µg of smooth LPS (O26:B6), Re
LPS and lipid A had been adsorbed onto microtiter wells, chromogenic
assay revealed 350 ± 29 ng (mean ± SE, n =
3) of smooth LPS, 325 ± 30 ng of Re LPS and 280 ± 18 ng of
lipid A were present in the wells after the washing procedures. Thus,
the solid phase ligands were similar in the amounts (by weight) bound
to the microtiter wells. Therefore, these results indicate the
different interactions of SP-A with smooth and rough LPS and further
demonstrate that the region of the neck plus CRD is directly involved
in the binding of SP-A to lipid A and rough LPS.
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Collectively, the results indicate that SP-A recognizes rough LPS but not smooth LPS in fluid phase as well as in solid phase.
The effect of SP-A upon LPS-induced TNF- expression in U937
cells
We next examined whether SP-A acted differently upon the cellular
responses to the distinct serotypes of LPS. The human macrophage-like
cell line, U937, was differentiated and incubated with LPS in the
absence or the presence of hSP-A for 2 h, and relative TNF-
mRNA levels were compared (Fig. 2A). Endotoxin-free hSP-A by
itself did not induce TNF- expression. When the cells were incubated
with 10 ng/ml smooth LPS (O26:B6) in the presence of 20 µg/ml hSP-A,
the TNF- mRNA level induced by smooth LPS was significantly
decreased. However, hSP-A failed to alter TNF- mRNA expression
induced by Re LPS. The secretion of TNF- into media was also
determined by L929 cell bioassay when differentiated U937 cells were
incubated with LPS for 5 h. Consistently, hSP-A dramatically
diminished TNF- secretion induced by smooth LPS but did not
attenuate Re LPS-stimulated TNF- secretion (Fig. 2B).
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secretion to the level of
8% of that induced by smooth LPS, the level of TNF- secretion in
the presence of heat-treated hSP-A was 107%. The results indicate that
heat treatment of SP-A completely destroys the inhibitory activity and
support the idea that the SP-A protein affects the cellular responses
to LPS.
The inhibitory effect of hSP-A upon smooth LPS-induced TNF-
secretion was clearly dependent upon the SP-A concentrations (Fig. 3A). Concentrations as low as
2 µg/ml hSP-A completely blocked TNF- secretion stimulated by 10
ng/ml smooth LPS. We also examined the effect of hSP-A upon various
concentrations of LPS (Fig. 3B). When 1–100 ng/ml smooth
LPS was co-incubated with 20 µg/ml hSP-A, TNF- secretion was
completely inhibited. However, the inhibitory effect of hSP-A upon very
high amounts of smooth LPS was apparently weak. hSP-A reduced TNF-
secretion stimulated by 1 µg/ml smooth LPS by only 40%. Increasing
the SP-A concentration up to 50 µg/ml did not alter the degree of the
inhibitory effects of SP-A. In contrast to smooth LPS, we found that 20
µg/ml hSP-A did not attenuate TNF- secretion stimulated by either
low (1 ng/ml) or high (1 µg/ml) concentration of Re LPS. These
results emphasize the different actions of SP-A upon the cellular
responses to distinct strains of LPS, and suggest the possibility that
SP-A may prevent CD14-dependent responses to smooth LPS because the
response to low but not high concentrations of LPS depends on CD14
(20, 32).
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SP-A does not bind smooth LPS but alters cellular response to
smooth LPS. Therefore, we speculated that the interaction of SP-A with
U937 cells might alter the cellular responses to LPS. This idea
initially led us to estimate the binding of SP-A to differentiated U937
cells. 125I-labeled SP-A was found to bind
differentiated U937 cells with high affinity (Fig. 4). The apparent
Kd calculated by Klotz plot was
2.8 x 10-9 M and the binding sites was
5.9 x 104 per cell, assuming a multimeric
molecular mass of 6.5 x 105 for SP-A (33).
The binding of SP-A to U937 cells was time-dependent, and its binding
reached saturation at 1 h at 37°C. When 1 µg/ml
125I-labeled SP-A was incubated with the cells,
50-fold excess unlabeled SP-A reduced
125I-labeled SP-A binding to the level of 5.5%
of control binding.
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secretion (Fig. 5). When the
cells were preincubated with hSP-A for 2 h and unbound hSP-A was
washed out, no TNF- was secreted by O26:B6 LPS added subsequently
(p < 0.002, compared with O26:B6 LPS alone
without pretreatment). This finding demonstrates that the inhibitory
effect of SP-A persists even after its removal from the medium. In
comparison with smooth LPS, TNF- secretion induced by rough (Re) LPS
was not attenuated by co-incubated hSP-A, and it was found that after
preincubating the cells with hSP-A before the addition of Re LPS,
TNF- secretion was significantly increased
(p < 0.01, compared with Re LPS alone without
pretreatment). To confirm that unbound SP-A was effectively removed
from the medium by the washing procedures, we determined the amount of
SP-A remaining in the medium. When differentiated U937 cells were
incubated with 20 µg/ml 125I-labeled SP-A,
washed, and replaced with medium that did not contain labeled SP-A,
only 0.5 ng/ml of 125I-labeled SP-A was
detectable in the medium. Therefore, it is unlikely that SP-A remaining
in the medium decreased smooth LPS-induced TNF- secretion, because
200 ng/ml of SP-A is required for the half-maximal inhibition (see Fig. 3
A). Taken together, the results support the idea that the
binding of SP-A to U937 cells alters the subsequent cellular responses
to LPS.
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Membrane CD14, a GPI-anchored membrane protein of 53–55 kDa, is
one of the identified receptors required for cellular responses to LPS.
We sought to isolate the SP-A binding proteins from differentiated U937
cells and determine whether CD14 is one of them. Cell surface proteins
on differentiated U937 cells were biotinylated, solubilized, and
applied to an affinity column covalently linked with hSP-A, and the
proteins binding to the affinity matrix were eluted in the presence of
EDTA. Several proteins were found to bind to the hSP-A affinity column
when the biotinylated proteins were visualized using avidin-HRP (Fig. 6, lane a). These proteins
were also analyzed by Western blot using mAb to human CD14. A protein
band with an apparent molecular mass of 55 kDa was detected (Fig. 6, lanes b and c). The results clearly demonstrate
that CD14 is one of the SP-A binding proteins on differentiated U937
cells.
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A, lane a), which
is in agreement with the previous report (34). For the ligand blot
analysis, the membrane was incubated with either hSP-A or BSA and
probed with anti-SP-A IgG. hSP-A that had bound to the membrane was
detected as a band corresponding to that of rsCD14 (Fig. 7A,
lanes b and c), demonstrating that hSP-A binds to
rsCD14. Furthermore, we examined the binding of hSP-A to rsCD14 coated
onto microtiter wells. hSP-A bound to rsCD14 in a
concentration-dependent manner (Fig. 7B). From these
results, we conclude that SP-A interacts directly with CD14 and that
the association of SP-A with CD14 does not require a GPI-anchor.
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For the purpose of clarifying the mechanism by which SP-A
modulates LPS-induced cellular responses, we then investigated whether
SP-A affects the interaction of CD14 with LPS. After rsCD14 was
preincubated with hSP-A for 1 h, the mixture of rsCD14 and hSP-A
was further incubated with smooth (O26:B6) or rough LPS coated onto the
wells, and the rsCD14 that bound to the solid phase LPS was detected
using anti-CD14 Ab. Preincubation of rsCD14 with hSP-A
significantly reduced the binding of rsCD14 to smooth LPS (Fig. 8). In contrast, the binding of rsCD14 to
Re LPS was increased by the preincubation of rsCD14 with hSP-A. The
results obtained from Fig. 8 are quite consistent with those
demonstrating that preincubation of U937 cells with SP-A decreases
cellular responses to smooth LPS but increases the responses to rough
LPS (see Fig. 5). Therefore, these data support the idea that the
principal mechanism of SP-A-mediated cellular responses is that the
direct interaction of SP-A with CD14 alters the subsequent binding of
LPS to CD14. Smooth LPS, which cannot bind to SP-A, may fail to
interact with CD14 when SP-A has bound to CD14, whereas rough LPS,
which binds to SP-A, may effectively associate with CD14 via an
SP-A-CD14 complex.
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expression in alveolar
macrophages
To confirm that the functions of SP-A examined with U937 cells
were relevant to alveolar environment, we investigated the effect of
SP-A on rat alveolar macrophages (Fig. 9). SP-A has been previously shown to
bind alveolar macrophages with high affinity (4). Consistently, hSP-A
exhibited a significant inhibitory effect on smooth LPS-induced TNF-
secretion by alveolar macrophages. By the preincubation of macrophages
with hSP-A, TNF- secretion induced by O26:B6 or O111:B4 was reduced
to 54.1 or 59.3% of that induced by LPS alone, respectively. In
contrast, hSP-A failed to inhibit rough LPS-stimulated cellular
responses. hSP-A did not affect Rc LPS-induced TNF- secretion and
dramatically increased Re LPS-induced TNF- secretion. These results
clearly demonstrate that SP-A modulates LPS-elicited responsiveness of
alveolar macrophages in the same manner as that of macrophage-like cell
line U937 cells. In addition, the rank order of TNF- secretion
(O26:B6, O111:B4 < Rc < Re) elicited by LPS in the presence
of SP-A appears to correlate well with that of the binding extent of
SP-A to LPS (see Fig. 1A). Taken together, these results
also support the idea that when SP-A has bound to CD14, SP-A-mediated
modulation of LPS-elicited responses may depend on the affinity between
SP-A and LPS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SP-A inhibited TNF- mRNA expression and TNF- secretion induced by
smooth LPS but not by rough LPS. It is unlikely that rough LPS acts
more potently than smooth LPS, because the concentration-dependent
stimulation by smooth and rough LPS generated almost equal amounts of
TNF- in differentiated U937 cells in this study. Furthermore, the
TNF- secretion stimulated by 100 ng/ml smooth LPS was completely
blocked by 20 µg/ml SP-A, while that by 1 ng/ml rough LPS was not
significantly inhibited. Approximately 2 mol equivalents of
octadecameric SP-A (33) are required to neutralize 1 mol equivalent of
smooth LPS in our assays. However, SP-A at molar ratio of 75:1 fails to
block rough LPS-stimulated TNF- secretion. Taken together, the
inhibitory effect of SP-A is quite restricted to smooth LPS-induced
cellular responses.
To understand the mechanism by which SP-A specifically prevents smooth
LPS-induced TNF- expression, several possible interactions were
considered. Clearly, SP-A does not affect the cellular responses by
binding directly to smooth LPS. The mode of action of SP-A is different
from MBP which has been shown to inhibit TNF- secretion induced by
streptococcal cell wall polysaccharides, rhamnose glucose polymers
(RGP), by its direct binding to RGP (37). Another possible mechanism
may be that SP-A interferes with the functions of the serum components
such as LPS binding protein (LBP) (38), sCD14 (22), and septin (39),
which have been known to accelerate LPS-induced cellular responses.
sCD14 and LBP are necessary for neutrophil responses to low
concentrations of smooth LPS but LBP alone is sufficient for response
to rough LPS (21). However, in the current study, the interaction of
SP-A with the serum components in media does not appear to be a primary
mechanism, because the inhibitory effect of SP-A persists even after
this protein is removed from the media. Cell-bound SP-A is quite
sufficient to modulate LPS-elicited cellular responses.
The present study clearly indicates that the interaction of SP-A with cells alters the cellular responses to LPS. Furthermore, we show that SP-A binds to CD14. Because SP-A does not associate with smooth LPS, it is reasonable to propose that the binding of SP-A to mCD14 may prevent smooth LPS from binding to mCD14, and consequently the smooth LPS-elicited cellular response is inhibited. This idea is clearly supported by the present results demonstrating that the binding of rsCD14 to smooth LPS is significantly decreased by the preincubation of rsCD14 with SP-A.
In contrast, the interpretation of the results obtained with SP-A and rough LPS seems more complex. Our data suggest that preincubation of SP-A with CD14 accelerates the binding of CD14 to rough LPS and increases rough LPS-mediated cellular responses. At present, we speculate that the interaction of rough LPS with CD14 is accelerated in the presence of SP-A which is a ligand for both rough LPS and CD14. The idea that SP-A can simultaneously cross-link with both rough LPS and CD14 is also supported by the results demonstrating that the domains of the SP-A molecule involved in the binding to rough LPS are different from those required for the binding to CD14 (H. Sano and Y. Kuroki, manuscript in preparation). Recent observations indicate that LPS which has bound to mCD14 activate the cells via subsequent interactions with unidentified transmembrane components (23, 40). Recently, human Toll-like receptor-2 and murine Toll-like receptor-4 each have been implicated in signaling by LPS and CD14 (24, 41, 42). An SP-A-rough LPS-mCD14 complex might effectively transfer rough LPS to another acceptor required for signaling. This idea may be analogous to the model of LBP-LPS-mCD14 ternary complex (43). Further studies are required to clarify the precise mechanism by which SP-A modulates the LPS signal transduction.
Two distinct proteins isolated from U937 cells have been identified as SP-A or C1q/MBL/SP-A receptor (44, 45), suggesting the existence of multiple receptors for SP-A. We also found several SP-A binding proteins isolated from solubilized U937 cells by SP-A affinity column, and further indicated that mCD14 was one of them. Isolation and investigation of the functions of unidentified SP-A receptors could also enable us to understand the precise mechanism of SP-A-mediated immune responses.
Although several serum molecules which affect LPS-induced cellular
responses have been well studied, little is known about the components
which exist in the alveolar liquid layer and modulate LPS-induced
responses of alveolar macrophages. Furthermore, although CD14 has been
well known as a major LPS receptor, the possible ligands for CD14 have
not been fully investigated. This study highlights that SP-A can bind
to CD14, and affects cellular responses to LPS in alveolar macrophages.
Because a number of Gram-negative bacteria colonizing the respiratory
tract do not express O-Ag or a part of the core oligosaccharides (17),
the interaction of SP-A with rough LPS is likely to be important within
the lung. The lipid A parts of E. coli and S.
minnesota examined in this study are similar to those of important
Gram-negative lung pathogens such as Pseudomonas aeruginosa
and Haemophilus influenzae, indicating that SP-A may also
interact with the clinical isolates of the pathogens which contain
rough LPS. The stark contrast of SP-A binding to distinct serotypes of
LPS is consistent with the recent findings that SP-A enhanced
phagocytosis of rough strain but not of smooth strain of bacteria by
macrophages (46). The interaction of SP-A with both CD14 and rough LPS
may also participate in the clearance of rough LPS, because the
internalization of LPS is also initiated by anchoring to mCD14 (43).
TNF- is a proinflammatory cytokine that plays a central role in many
human diseases and it exhibits both beneficial and pathological
effects. Overproduction of TNF- by LPS-activated macrophages could
potentially worsen the diseased state. Thus, SP-A-mediated
down-regulation of TNF- expression induced by smooth LPS may play an
important dampening role in controlling inflammation.
In this study lipid-free SP-A inhibits smooth LPS-elicited cellular responses. Because SP-A interacts with lipids and is thought to exist as a lipoprotein in the alveoli, it is important to determine whether a similar effect of SP-A can be seen in the presence of surfactant lipids. We are now investigating the effects of hydrophobic surfactant components upon the response of U937 cells to LPS and SP-A.
The lungs appear to remain in a relatively uninflamed state despite the
daily exposure to ambient air that contains significant numbers of
microorganisms and their derivative products such as LPS. Expression of
SP-A is enhanced in response to intratracheal instillation of smooth
LPS (47, 48), indicating that the lung is highly resistant to the
pathological effects of LPS. However, neutrophils and macrophages
removed from the alveolar environment are exquisitely sensitive to LPS.
One idea emerging from this study is that SP-A functions under ordinary
conditions as a potent anti-inflammatory agent either sequestering
microorganisms or their products or effecting an anti-inflammatory
response on the target cells. On the other hand, SP-A concentrations in
bronchoalveolar lavage fluids decrease in patients with acute lung
injury (49) and bacterial pneumonia (50). It is unclear why the
different results are obtained in humans and animal models. This may
implicate complicated responses in the disease states of human. The
reduction of SP-A concentrations in human alveolar fluids may also be a
consequence of consumption of SP-A that has interacted with bacteria
and their derivatives. Another interpretation is that TNF- released
by activated macrophages in patients with acute lung injury and
pneumonia might reduce SP-A levels because TNF- suppresses SP-A
synthesis in type II cell-like cell lines (51).
In conclusion, this study demonstrates that SP-A exhibits different interactions with distinct serotypes of LPS and affects differently their elicited cellular responses. We propose that the modulation of LPS-induced cellular responses by SP-A occurs by the direct interaction of SP-A with CD14.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshio Kuroki, Department of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060, Japan. E-mail address:
3 Abbreviations used in this paper: SP-A, surfactant protein A; hSP-A, human SP-A; CRF, collagenase-resistant fragment; CRD, carbohydrate recognition domain; SP-D, surfactant protein D; MBP, mannose binding protein; LBP, LPS binding protein; mCD14, membrane CD14; sCD14, soluble CD14; rsCD14, recombinant soluble CD14, PVDF, polyvinylidene difluoride; Re LPS, LPS derived from S. minesota strain Re 595 (Re mutant); RcLPS, LPS derived from E. coli strain J5 (Rc mutant).
Received for publication December 1, 1998. Accepted for publication April 22, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibits expression of pulmonary surfactant
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), Re LPS (
), Rc (J5) LPS (•), E.
coli O26:B6 (
), or O111:B4 (
) was coated onto microtiter
wells and incubated with the indicated concentrations of hSP-A
(A) or its CRF (B) for 5 h at
37°C. The binding of the proteins to lipids was detected using
anti-human SP-A IgG as described under Materials and
Methods. The data shown are mean ± SE of three
experiments. *, p < 0.02; **,
p < 0.05 when compared with the binding of SP-A to
011:B4.
) µg/ml of hSP-A for
5 h. TNF-
