1 Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710; and 2 Department of Anesthesiology, Hannover Medical School, 30625 Hannover, Germany
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ABSTRACT |
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Surfactant protein (SP) A and SP-D are involved in multiple immunomodulatory functions of innate host defense partly via their interaction with alveolar macrophages (AMs). In addition, both SP-A and SP-D bind to bacterial lipopolysaccharide (LPS). To investigate the functional significance of this interaction, we first tested the ability of SP-A and SP-D to enhance the binding of tritium-labeled Escherichia coli LPS to AMs. In contrast to SP-D, SP-A enhanced the binding of LPS by AMs in a time-, temperature-, and concentration-dependent manner. Coincubation with surfactant-like lipids did not affect the SP-A-mediated enhancement of LPS binding. At SP-A-to-LPS molar ratios of 1:2-1:3, the LPS binding by AMs reached 270% of control values. Second, we investigated the role of SP-A in regulating the degradation of LPS by AMs. In the presence of SP-A, deacylation of LPS by AMs increased by ~2.3-fold. Pretreatment of AMs with phosphatidylinositol-specific phospholipase C had no effect on the SP-A-enhanced LPS binding but did reduce the amount of serum-enhanced LPS binding by 50%, suggesting that a cell surface molecule distinct from CD14 mediates the effect of SP-A. Together the results for the first time provide direct evidence that SP-A enhances LPS binding and degradation by AMs.
collectins; Escherichia coli lipopolysaccharide
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INTRODUCTION |
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PULMONARY SURFACTANT PROTEIN (SP) A and SP-D belong to a group of collagen-like C-type or Ca2+-dependent mammalian lectins called collectins. These hybrid molecules are oligomers of trimeric subunits comprising three polypeptide chains, each having as major regions a glycosylated collagenous domain and a globular COOH-terminal (lectin) carbohydrate recognition domain (CRD) (8, 45). The collagenous domains have been reported to be ligands for receptors on phagocytes and, for some collectins, mediate activation of the classic complement pathway. The CRD participates in a selective, Ca2+-dependent recognition of oligosaccharide configurations on bacterial and viral surfaces (12, 35).
Plasma C-type collectins such as mannose-binding protein, conglutinin, and collectin-43 have been implicated in the innate, immunoglobulin-independent immune defense system. For example, mannose-binding protein, an acute-phase human serum lectin structurally homologous to SP-A, acts as an opsonin for a variety of microorganisms (29, 44), and can substitute for C1q in triggering the complement pathway (23). Conglutinin, a bovine serum lectin that shares structural similarity and a high degree of amino acid sequence identity with SP-D, has also been found to have both antibacterial (10) and antiviral activities (13) and to bind to gp160, a glycoprotein of human immunodeficiency virus-1 (1) and to glycoproteins of the complement system (21).
Because of their structural similarity with plasma C-type collectins, SP-A and SP-D have been proposed to have similar functions in the alveolar space. Indeed, SP-A (38) and SP-D (18, 32) have been reported to bind in a specific, Ca2+-dependent manner to alveolar macrophages (AMs), the pivotal lung cell responsible for both nonantibody-mediated host defense and clearance of SP-A (53) and SP-D (7). In addition, both SP-A (27, 37, 46, 49) and SP-D (19, 22, 25, 34, 37) bind to and stimulate the uptake of a variety of pathogens by AMs, suggesting an enhancing effect on the clearance of pathogens entering the lung.
In acute bacterial pneumonia and sepsis-induced acute respiratory distress syndrome (ARDS), lipopolysaccharide (LPS) amplification pathways (28) are considered responsible for controlling pulmonary proinflammatory activity. LPS, the biologically active constituent of gram-negative bacteria, consists of lipid A, which is of central significance in cell activation and induction of proinflammatory responses, a core oligosaccharide, and a terminal polysaccharide of variable composition that determines the bacterial serotype. The lipid A moiety of LPS that expresses full endotoxic activity contains a glucosamine disaccharide with two phosphoryl groups, six covalently linked fatty acids, and nonhydroxylated fatty acids that substitute for hydroxyl groups to form acyloxyacyl groups (40, 43). This structural feature is required for responses to LPS by monocytes, macrophages, endothelial cells, and neutrophils, i.e., the production of bioactive lipids, reactive oxygen species, and a variety of cytokines. Enzymatic deacylation (removal of secondary acyl chains from the lipid A moiety) of LPS by phagocytic cells (33) has a potential LPS-detoxifying role because deacylated LPS is substantially less active at stimulating proinflammatory cell responses (16, 17).
SP-A and SP-D interact with LPS of various phenotypes (15, 19, 48). SP-A binds to rough LPS from Salmonella minnesota and Escherichia coli (48) as well as to smooth LPS (15). SP-D binds to rough LPS of E. coli, S. minnesota, Klebsiella pneumoniae, and Pseudomonas aeruginosa (19, 22). Several lines of evidence suggest that SP-A and SP-D may play an important role in the response of the lung to LPS. For example, LPS-induced alterations in SP-A and SP-D metabolism in vivo are suggested by increased protein and mRNA concentrations as an early response to intratracheally administered LPS in rats (31, 42, 47, 50). In addition, specific alterations of the biological activities of SP-A in vitro occur in the presence of LPS: SP-A binding to AMs increases (4), SP-A-enhanced phagocytosis of bacteria by AMs decreases (15), and tumor necrosis factor release by LPS-stimulated AMs is inhibited by SP-A (30).
In the present study, we focused our attention on the impact of SP-A and SP-D on LPS clearance by AMs. We first investigated whether SP-A and SP-D contribute to the uptake/binding of 3H-labeled E. coli LPS by AMs. Because SP-A, in contrast to SP-D, increased LPS uptake/binding, we subsequently tested whether SP-A also contributes to LPS deacylation by AMs. Third, we evaluated the role of membrane-bound CD14 (mCD14) in SP-A-increased LPS binding. mCD14 is a cell surface receptor on phagocytes that rapidly binds LPS complexed to the serum LPS-binding protein (LBP) (11, 54).
These studies provide, for the first time, direct evidence of SP-A-enhanced uptake/binding of LPS by AMs and suggest that a receptor distinct from mCD14 may mediate this effect. In addition, we show that SP-A enhances the deacylation of E. coli LPS by AMs.
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MATERIALS AND METHODS |
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Reagents. Radiolabeled LPS from the
LCD 25 strain of E. coli K12 was
obtained from List Biological Laboratories (Campbell, CA). The
E. coli LPS was an Rb chemotype, with
the tritium label incorporated into the fatty acyl chains of the lipid
A moiety of the molecule. The specific activity of the
E. coli LPS preparation was 2.08 × 106 dpm/µg. CytoScint
liquid scintillation cocktail was from ICN (Costa Mesa, CA). Lipids
(L--dipalmitoylphosphatidylglycerol, L-
-dipalmitoylphosphatidylcholine,
egg phosphatidylcholine, and cholesterol) were purchased from Avanti
Polar Lipids (Birmingham, AL). Ethyl alcohol was from USP Aaper Alcohol
and Chemical (Shelbyville, KY). Triton X-100 and bovine serum albumin
(BSA) reported to have endotoxin levels < 0.1 ng/mg were from Sigma
(St. Louis, MO). Dulbecco's phosphate-buffered saline (DPBS) and fetal
bovine serum were obtained from GIBCO BRL (Grand Island, NY).
Phosphatidylinositol-specific phospholipase C (PI-PLC) from
Bacillus cereus was from Boehringer Mannheim (Indianapolis, IN).
Purification of SP-A. SP-A was isolated from lung lavage fluid from patients with alveolar proteinosis as previously described in detail (52). Briefly, the whole surfactant pellet from lung lavage fluids was extracted with butanol; butanol-insoluble proteins were resolubilized with octylglucopyranoside (OGP); and subsequently, SP-A was solubilized in 5 mM Tris-buffered water, pH 7.4. Residual OGP was removed by dialysis against 5 mM Tris-buffered water, pH 7.4. To remove endotoxin, SP-A was treated with OGP and polymyxin B agarose beads as previously described in detail (30). SP-A and SP-D preparations were tested for bacterial endotoxin with a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). All SP-A preparations used contained <0.02 pg endotoxin/µg SP-A. All SP-D preparations used contained <0.6 pg endotoxin/µg SP-D.
Purification of SP-D. SP-D was isolated from lung lavage fluids of silica-treated rats as previously described (6). Briefly, to induce surfactant accumulation, anesthetized rats received 25 mg of silica in 0.5 ml of saline intratracheally and were killed by exsanguination ~4 wk later, and their lungs were lavaged six times with 150 mM NaCl and 5 mM Tris, pH 7.4. Lavage samples were centrifuged at ~27,000 g for 30 min at 10°C. The supernatant was mixed with maltose-Sepharose (9) and incubated for 18 h at 4°C. The slurry was centrifuged at 2,400 g for 15 min at 10°C. The supernatant was removed, and the slurry was washed seven times. The slurry was then resuspended in 20 mM Tris, 150 mM NaCl, and 5 mM CaCl2, pH 7.8; incubated for 30 min at 4°C; and centrifuged at 2,400 g for 15 min. After the final wash, the supernatant was removed, and the maltose-Sepharose was resuspended in 50 mM Tris, 150 mM NaCl, and 10 mM EDTA, pH 7.4. The Ca2+-dependent maltose-binding proteins were eluted from the beads by washing them three to four times. The fractions containing the highest SP-D concentrations were pooled and further purified by gel filtration (36).
Isolation of cells. AMs were isolated by lung lavage of male, specific pathogen-free Sprague-Dawley rats weighing 250-300 g (Charles River Laboratories, Raleigh, NC). In brief, the rats were anesthetized with pentobarbital sodium and killed by exsanguination. The lungs were removed and lavaged eight times with warmed (37°C) DPBS containing 0.2 mM EDTA. Lavage samples were centrifuged at 200 g for 10 min. Cell recovery routinely averaged 3-8 × 106 cells/animal. The viability of the cells was determined by erythrosin B exclusion and averaged 95-97%.
Preparation of liposomes. Small
unilamellar liposomes composed of surfactant-like lipids were prepared
by extrusion from a French pressure cell as previously described (53).
The liposomes consisted of (by weight) 52%
L--dipalmitoylphosphatidylcholine, 26% egg phosphatidylcholine, 15%
L-
-dipalmitoylphosphatidylglycerol, and 7% cholesterol.
Incubation conditions. All microfuge tubes were incubated overnight at 4°C with 1% low-endotoxin BSA in DPBS and then washed two times with water. Isolated AMs (5-5.5 × 106) were resuspended into 1 ml of incubation buffer (DPBS with 0.9 mM CaCl2 and 0.1% BSA). E. coli [3H]LPS plus SP-D or SP-A in varying amounts was added to the cell suspension. The cells were incubated with gentle shaking at 4 or 37°C for various amounts of time. After incubation, the cells were collected by centrifugation at 200 g for 10 min, and the medium was saved for further measurement. The resuspended cells were washed, transferred to a new tube to minimize nonspecific adsorption of radioactivity to the tube, and washed again two times by centrifugation. The earliest time point was obtained by centrifugation and washing immediately after the addition of E. coli [3H]LPS or E. coli [3H]LPS plus SP-A. The elapsed time before the beginning of the first centrifugation was ~20 s. The final cell pellet was resuspended in 0.2 ml of incubation buffer, and 0.1 ml was transferred to a scintillation vial. Four milliliters of scintillation fluid were added to each vial, and the radioactivity was measured in a LS 1800 scintillation counter (Beckman, Fullerton, CA). Aliquots of the medium (0.1 ml) were analyzed by scintillation counting in exactly the same way.
To investigate the effect of surfactant-like lipids on E. coli [3H]LPS binding by AMs, liposomes at a final concentration of 20 µg phospholipid/ml were coincubated in additional experiments.
The term "uptake/binding" is used to refer to the association of E. coli [3H]LPS with the cell and is a measure of both membrane-bound and internalized LPS (17).
Preparation of LPS. The lyophilized E. coli LPS product was reconstituted at a concentration of 1 µg/µl in 0.1% triethylamine and 10 mM Tris, pH 8. The stock solution was diluted to 0.02 µg/µl in PBS containing 0.1% low-endotoxin BSA.
Ethanol extraction of lipids. To study
the metabolism and degradation of LPS, the ethanol precipitation method
described by Luchi and Munford (24) was used. Isolated AMs (5 × 106/ml) were incubated
with [3H]LPS alone or
[3H]LPS plus 5 µg/ml
of SP-A at 37°C for 60 min. After centrifugation, the cells were
washed, transferred to new tubes, washed again two times to remove
extracellular [3H]LPS,
and finally reincubated at 37°C for 120 min. After centrifugation, 100 µl of Triton X-100 and BSA (final concentrations 1.25% Triton X-100 and 2.5 mg/ml of BSA) were added to 100-µl aliquots of the supernatants, 1 ml of 95% ethanol was added, and the samples were chilled overnight at 20°C. The cells were resuspended in
DPBS, transferred to new tubes, and washed again. Finally, the cells were lysed in 0.1% Triton X-100 in DPBS, and 25-µl aliquots were counted. One hundred-microliter aliquots of resuspended cells were
extracted with 100 µl of Triton X-100 and BSA, followed by 1 ml of
95% ethanol, and the samples were chilled overnight at
20°C. Finally, tubes were centrifuged at 9,000 g for 15 min at 4°C. The
supernatants were carefully removed, and 100-µl aliquots were
transferred to scintillation vials. The pellets were resuspended with
0.1% Triton X-100 in DPBS, and 100-µl aliquots were transferred to
scintillation vials. Four milliliters of scintillation fluid were added
to each vial. The 3H content of
the supernatants and pellets was measured by scintillation counting.
Control experiments included cell-free
[3H]LPS and
[3H]LPS plus SP-A incubations.
PI-PLC pretreatment of AMs. AMs were incubated for 60 min at 37°C in Ca2+- and Mg2+-free DBPS containing 0.1% BSA and 2.5 U/ml of PI-PLC. After this treatment, the cells were resuspended in cold DBPS, washed, transferred to new tubes, and washed again by centrifugation. The binding assay described in Incubation conditions was carried out for 120 min at 4°C to minimize recycling of mCD14 to the cell surface. Control experiments included untreated cells.
Statistical analysis. Data were analyzed with the Mann-Whitney U-test or Student's t-test for unpaired samples. P < 0.05 was considered significant.
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RESULTS |
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Effects of SP-A and SP-D on the uptake/binding of E. coli
[3H]LPS by AMs.
As shown in Fig. 1, SP-A enhanced the
uptake/binding of E. coli
[3H]LPS by AMs
incubated at 37°C for 120 min. In the presence of 2 µg SP-A /ml,
the uptake/binding of E. coli
[3H]LPS by AMs was
increased by ~170 ± 9% of control (no-SP-A or -SP-D) value
(P < 0.004). In contrast, 2 µg
SP-D/ml did not significantly increase the amount of bound
E. coli
[3H]LPS (108 ± 4%
of control value).
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Concentration, time, and temperature dependency of SP-A-enhanced
uptake/binding of E. coli
[3H]LPS by AMs.
The enhancing effects of SP-A on the uptake/binding of
E. coli
[3H]LPS by AMs were
concentration dependent (Fig. 2). Maximal
enhancement was observed in the presence of 5-10 µg/ml of SP-A,
which resulted in a binding by AMs of >200% of control (no-SP-A)
value.
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Effects of surfactant-like lipids on the uptake/binding of E. coli
[3H]LPS by AMs.
As shown in Fig. 5, the SP-A-enhanced
amount of bound E. coli
[3H]LPS was similar in
the presence and absence of liposomes made of surfactant-like lipids
(20 µg/ml).
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Effects of E. coli [3H]LPS
concentration on SP-A- enhanced E. coli
[3H]LPS uptake/binding by AMs.
As shown in Fig. 6, the amount of bound
E. coli
[3H]LPS increased with
increasing amounts of E. coli
[3H]LPS added. The
cellular binding sites for LPS were apparently not saturated within the
LPS concentrations used. SP-A-to-LPS molar ratios of ~1:2-1:3
resulted in maximal SP-A-enhanced LPS binding (270% of control value).
There was no significant sedimentation of the E. coli
[3H]LPS in the absence
of cells (<0.2%).
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Effects of SP-A on the deacylation of E. coli
[3H]LPS by AMs.
Because the 3H label was confined
to the fatty acyl chains located in the lipid A moiety of the
E. coli
[3H]LPS used in our
investigations, the deacylation of LPS was measured by ethanol
precipitation and subsequent scintillation counting of the marker in
the ethanol-soluble phase. As shown in Fig.
7, the SP-A-enhanced binding of
E. coli
[3H]LPS by AMs was
accompanied by an increased deacylation of E. coli
[3H]LPS. In the
presence of SP-A, the percentage of the total cell-associated 3H radioactivity that was ethanol
soluble after 120 min of incubation was 7.4 ± 1.2% compared with
3.2 ± 0.4% in the absence of SP-A (P < 0.05).
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Effects of PI-PLC pretreatment of AMs on E. coli
[3H]LPS binding by AMs in the
presence of SP-A or serum at 4°C.
mCD14, a glycosylphosphatidylinositol (GPI)-linked 55-kDa protein
present on the surface of macrophages, monocytes, and polymorphonuclear neutrophils (PMNs), rapidly binds LPS in the presence of LBP, a
component of serum, as part of a multicomponent receptor (11, 14, 54,
55). Treatment of cells with PI-PLC removes GPI-anchored mCD14 from the
outer cell membrane (14, 54). To determine whether mCD14 may be
involved in the SP-A-mediated enhancement of LPS binding, we treated
AMs with PI-PLC before incubation with SP-A. As shown in Fig.
8, PI-PLC pretreatment of cells reduced the
cell-associated E. coli
[3H]LPS by ~50% in
the presence of serum, whereas it had no effect in the presence of
SP-A. There was no significant sedimentation of E. coli
[3H]LPS in the absence
of cells (<0.03%).
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DISCUSSION |
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Our primary goal in this investigation was to determine whether SP-A and SP-D, components of the pulmonary innate immune system (51), enhance the uptake/binding of E. coli LPS by AMs, the primary host defense cell in the noninflamed lung. The results show that SP-A, but not SP-D, enhances the uptake/binding of E. coli LPS by AMs in a concentration-, time-, and temperature-dependent manner. In addition, the SP-A-enhanced uptake/binding of E. coli LPS by AMs is similar in the presence and absence of surfactant-like lipids.
We found maximal SP-A-enhanced E. coli [3H]LPS binding by AMs at SP-A concentrations comparable to the estimated physiological concentration of ~4-11 µg/ml, based on a calculated SP-A concentration of ~360 µg/ml in the hypophase of the rat lung (41) and a portion of 1-3% of SP-A that is not bound to surfactant phospholipids (2). Notably, three different preparations of both SP-A and SP-D were used for these experiments. We used LPS concentrations well within the range of bronchoalveolar lavage LPS levels found in patients with pneumonia (up to 300 ng/ml) (28), beginning with LPS concentrations at the threshold for serum-free LPS-induced AM stimulation (10 ng/ml). Assuming molecular masses of 630 and 4 kDa for SP-A and LPS, respectively, SP-A-to-LPS molar ratios of ~1:2-1:3 resulted in maximal SP-A-enhanced binding.
We investigated the effects of surfactant lipids on SP-A-mediated LPS binding for two reasons. First, the majority but not all of the SP-A in lavage fluid is bound to lipid (2). Second, AMs internalize and degrade surfactant lipids and SP-A and contribute significantly to the clearance of both of them, with SP-A enhancing the uptake of surfactant lipids by AMs in a time-, temperature-, and concentration-dependent manner (53). Our results indicate that the SP-A-enhanced binding of E. coli [3H]LPS by AMs is similar in the presence and absence of liposomes, consistent with previous observations that surfactant-like lipids do not affect LPS binding to SP-A (15). Thus these results suggest that SP-A can mediate LPS binding in both the presence and absence of lipids.
The mechanism of the SP-A-mediated enhancement of LPS uptake/binding is not known. Both SP-A (15, 48) and SP-D (19, 22) bind to rough LPS of various phenotypes. Bacteria with rough LPS phenotypes that are deficient in O-polysaccharide, and fragments of the core oligosaccharide are most common among bacteria that colonize the surfaces of the upper aerodigestive tract (40). SP-A may bind to bacterial LPS via interaction with the lipid A moiety (15, 48), whereas SP-D may interact with the inner core oligosaccharides of the LPS molecule (19). The SP-A domain that interacts with LPS is not known, but the lipid-binding region of SP-A is thought to be a possible LPS-binding domain (48). SP-D appears to interact with rough LPS via its CRD (19). The E. coli LPS used in our investigations is an Rb chemotype containing core terminal glucose and heptose residues that are strongly assumed to mediate SP-D-LPS binding (19). In fact, both SP-A and SP-D, which were biotinylated, bound to the Rb LCD 25 strain of E. coli LPS in microtiter plate binding assays (data not shown). Therefore, our data suggest that binding of LPS to SP-D is not sufficient to stimulate LPS uptake by AMs.
The cell surface receptor involved in the SP-A-mediated enhancement of
LPS uptake/binding is not known. SP-A-LPS complexes may bind to AMs via
one of the putative SP-A receptors (5, 26). It has been shown that the
binding affinity of SP-A to AMs is much higher (dissociation constant = 4 × 109 M) (38) than
that of SP-D (dissociation constant = 1.4 × 10
6 M) (18), and most
probably, SP-A binding occurs via a different binding domain than its
CRD. The fact that SP-D binds to AMs as well as to LPS via its CRD may
be another explanation for the different ability of the two collectins
to affect LPS uptake/binding.
Our data suggest that mCD14, the major LPS-binding cell surface receptor on phagocytes, does not mediate the effect of SP-A. In the presence of LBP, an acute-phase serum protein, mCD14 rapidly binds LPS, and the sensitivity of cells to low concentrations of LPS is greatly increased (11, 54). In our studies, bovine serum increased LPS binding by AMs by a factor of two, which is consistent with its effect on LPS binding by PMNs (24). In addition, PI-PLC pretreatment of AMs, which has been shown to reduce GPI-anchored mCD14 on immune cells (14, 54), reduced the amount of cell-associated LPS by ~50% in the presence of serum, which contains LBP (3). In contrast, the SP-A-mediated enhancement of LPS binding was not altered after PI-PLC pretreatment of the cells, suggesting that mCD14 is not mediating the effect of SP-A. However, because anti-rat CD14 antibodies are not commercially available, to the best of our knowledge, the conclusion that the effect of SP-A is CD14 independent is based exclusively on the striking difference in LPS binding by PI-PLC-pretreated cells in the presence of either serum or SP-A.
Our studies show that SP-A enhances the deacylation of LPS by AMs. Approximately 7.4% of the cell-associated 3H radioactivity was ethanol soluble after 120 min of incubation, a rate of ~3%/h. Because the enzyme acyloxyacyl hydrolase removes only two of the six acyl chains from the lipid A moiety of LPS (24), the release of 3%/h of the 3H radioactivity might represent a rate of deacylation of 9-18%/h of LPS molecules (due to release of one acyl chain from 18% of the LPS molecules or release of two acyl chains from 9% of the molecules). This rate of deacylation is comparable to the reported rate of deacylation of [3H]LPS by neutrophils of 9-18%/h (24).
The functional consequences of the SP-A-mediated enhanced binding of
LPS to AMs are not known and are likely to be complex. Conceivably,
either an augmentation or an inhibition of macrophage activation and
production of cytokines and other inflammatory mediators could result
from the effect of SP-A. For example, the SP-A-mediated increase in LPS
binding could result in enhanced signaling and production of
proinflammatory cytokines if SP-A has an effect similar to LBP, which,
at low concentrations, increases cytokine production in the presence of
LPS (54). Interestingly, high concentrations of LBP inhibit LPS-induced
proinflammatory effects (20). Alternatively, because SP-A enhanced the
deacylation of LPS and because deacylated LPS is less proinflammatory
than intact LPS (33, 39), the net effect could be an inhibition of
LPS-induced cell responses. A previous study from our laboratory (30)
showed that SP-A inhibits tumor necrosis factor- production by
LPS-stimulated AMs. Because partially deacylated LPS can inhibit LPS-induced cytokine release (16, 17), these results suggest that,
under certain conditions, the SP-A-enhanced deacylation of LPS may
result in a decrease in cytokine production. It seems that the
consequences of the SP-A-enhanced binding of LPS are likely to be
influenced by many variables including the concentration of both SP-A
and LPS, the time course over which the LPS exposure occurs, the state
of activation of the immune cells, and the relative extent and rate at
which binding and deacylation occur.
If, in fact, SP-A acts to reduce the proinflammatory effects of LPS, then in the normal lung where SP-A levels are high, it may act to enhance LPS clearance and detoxification. In contrast, in lung diseases including ARDS, acute lung injury, and pulmonary bacterial infections, in which bronchoalveolar lavage levels of SP-A have been reported to be decreased (reviewed in Ref. 51), the lack of SP-A may allow for LPS-induced cell activation via other pathways. For example, it has been demonstrated in ARDS patients that the levels of bronchoalveolar LBP and soluble CD14 are elevated and strongly correlate with total lavage protein and PMN infiltration, two measures of lung inflammation (28). Thus when pulmonary SP-A concentrations are lower than normal and CD14 and LBP levels are increased in the lungs, LPS may induce proinflammatory consequences, further contributing to the acute lung injury. Future investigations will be required to elucidate the mechanisms and consequences of SP-A-mediated binding of LPS to immune cells.
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ACKNOWLEDGEMENTS |
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We thank Sabrena Mervin-Blake and Julie Taylor for excellent technical assistance, Dr. Qun Dong for helpful suggestions with the phosphatidylinisitol-specific phospholipase C assay, and Dr. Stephen Young for critical review of the manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-51134.
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 and other correspondence: J. R. Wright, Box 3709, Dept. of Cell Biology, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: J.Wright{at}cellbio.duke.edu).
Received 17 August 1998; accepted in final form 1 December 1998.
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