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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 353-360, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4812

Surfactant Protein A Inhibits Lipopolysaccharide-Induced Immune Cell Activation by Preventing the Interaction of Lipopolysaccharide with Lipopolysaccharide-Binding Protein

Cordula Stamme, Mareike Müller, Lutz Hamann, Thomas Gutsmann and Ulrich Seydel

Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Bioscience, Borstel; Department of Anesthesiology, University Hospital Lübeck, Lübeck; Department of Immunology and Cell Biology, Research Center Borstel, Borstel; and Department of Microbiology and Hygiene, Charité Medical Center, Humboldt University Berlin, Berlin, Germany

Address correspondence to: Dr. Cordula Stamme, Dept. of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany. E-mail: cstamme{at}fz-borstel.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pulmonary surfactant protein (SP)-A, an innate immune molecule, modifies lipopolysaccharide (LPS)-induced cell responses. Because SP-A avidly binds to the deep rough (Re) mutant of LPS, we first investigated the functional consequences of this interaction and found that preincubation of Re-LPS with SP-A significantly and in a dose-dependent manner decreased the sensitivity of rat alveolar macrophages and human mononuclear cells to Re-LPS–induced activation at limited amounts of LPS-binding protein (LBP). At high LBP concentrations, the SP-A–mediated cellular inhibition of Re-LPS–induced activation was abrogated. Because LBP-catalyzed binding of LPS to CD14 is essential for low-dose LPS-induced signaling, we then hypothesized that SP-A inhibits Re-LPS–induced immune cell activation via inhibiting the binding of Re-LPS to LBP. Binding competition experiments employing a surface plasmon resonance technique showed that Re-LPS preincubated with SP-A bound to LBP to a significantly lesser extent than Re-LPS alone. For enhanced cellular association of [3H]LPS/SP-A complexes to occur, the expression of membrane-bound CD14 by human embryonic kidney cells 293 was not essential. Therefore, the ability of SP-A to inhibit immune cell activation by Re-LPS may be due to its ability to block the binding of Re-LPS to LBP and prevent the initiation of the LBP/CD14 pathway for inflammatory reactions in the lung.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • Dulbecco's phosphate-buffered saline, DPBS • electrophoretic mobility shift assay, EMSA • fetal calf serum, FCS • human embryonic kidney, HEK • lipopolysaccharide, LPS • LPS-binding protein, LBP • membrane-bound CD14, mCD14 • nuclear factor {kappa}B, NF-{kappa}B • phosphatidylserine, PS • Rb mutant of Escherichia coli strain LCD 25 LPS, Rb-LPS • Re mutant of Escherichia coli strain F515, Re-LPS • recombinant human, rh • soluble CD14, sCD14 • surfactant protein, SP • surface plasmon resonance, SPR • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacterial lipopolysaccharides (LPS) represent highly potent stimuli of mammalian immune cells to express a series of genes coding for immunoregulatory, inflammatory, and cytotoxic molecules. In human sepsis or systemic inflammatory response syndrome, exaggerated responses to LPS may result in multiorgan failure and shock (1). Gram-negative bacteria express either smooth (S-form or wild-type) LPS or rough (R-form) LPS mutants. S-LPS is composed of O-antigen, complete core oligosaccharides, and lipid A. All mutant LPS, classified as Ra, Rb, Rc, Rd, and Re chemotypes, lack O-antigen, but possess lipid A and progressively shorter core oligosaccharides (2). LPS carrying only the 2-keto-3-deoxyoctulosonic acid units bound to the lipid A domain are classified as deep rough (heptoseless) mutant (Re) LPS (2, 3). Bacteria with R-LPS phenotypes are most common among pathogens that colonize the upper aerodigestive tract (3).

The mechanism(s) by which LPS activates immune cells have been elucidated in some detail (4). At least four proteins are used by myeloid cells to mount a sensitive response to LPS: LPS-binding protein (LBP), CD14, Toll-like receptor (TLR) 4, and MD-2 (5–9). LBP binds to the lipid A domain of LPS (5, 6), catalyzes the binding of LPS to soluble (s) and membrane-bound (m) CD14, and enhances CD14-mediated cell activation by increasing the affinity of binding of CD14 to LPS by yielding two KDs with significantly higher affinity (7). TLR-4 (8), MD-2 (9), and ion channels (10) have been identified as the postulated proteins that act in concert with the CD14/LPS receptor complex in efficient transmembrane signaling.

Pulmonary surfactant protein (SP)-A, the most abundant of the surfactant proteins, belongs to a group of collagenous carbohydrate-binding proteins known as collectins that mediate a variety of immune cell functions in vitro and in vivo (11–15). SP-A interacts with a wide range of pathogens, shows specific interaction with macrophages, and modulates the function of phagocytic cells (13). The inflammatory response to a variety of pathogens in SP-A–deficient mice are consistent with an anti-inflammatory effect of SP-A in vivo (14, 15, 24). These findings strongly support the idea that SP-A plays an important role in the innate, nonclonal defense system of the lung.

A number of studies have shown that SP-A modulates immune cell activation induced by both S- and R-LPS in vitro (16–23). Importantly, SP-A–deficient mice exhibit increased lung inflammation following intratracheal challenge to S-LPS (24). However, the mechanisms of SP-A–mediated modulation of LPS-induced activation have not been fully elucidated. SP-A binds to the lipid A domain of the R-LPS molecule via its lectin domain (16, 25, 26). The neck domain of SP-A directly interacts with the protein backbone of CD14 (26). Direct modulation of LPS-induced immune cell activation through interaction of SP-A with mCD14 has been proposed (20, 26). In addition, indirect modulation of cellular responses through an altered, SP-A–mediated presentation of R-LPS to the cells may occur.

Besides their well-known function as serum constituents, LBP and sCD14 are present in the normal alveolar fluid (27), and mCD14 mediates the responses of alveolar macrophages to LPS in vitro (27). LBP expression can be induced in vitro by pulmonary type II epithelial cells (28). In vivo, the concentrations of LBP and sCD14 in bronchoalveolar fluid rise by ~ 10-fold in patients with acute lung injury (29), suggesting that the local production of LBP in the lung may contribute significantly to host defense to LPS (30).

In the present study, we focused our attention on the impact of Re-LPS/SP-A interaction on immune cell function. Because the preincubation of Re-LPS with SP-A dramatically decreased the LBP-dependent sensitivity of macrophages and monocytes to Re-LPS, we subsequently tested whether both proteins, SP-A and LBP, depending on their local concentration, compete for Re-LPS binding. To test this hypothesis, we used a surface plasmon resonance technique as a binding assay to detect interactions of LBP, Re-LPS, and Re-LPS preincubated with SP-A with immobilized phospholipid matrices. We found that preincubation of Re-LPS with SP-A almost completely inhibited the subsequent binding of Re-LPS to LBP. In addition, the SP-A–mediated enhancement of [3H]Rb-LPS uptake by human embryonic kidney (HEK)293 cells was independent of the expression of mCD14, and SP-A did not promote but rather inhibited the serum-enhanced interaction of [3H]Rb-LPS with mCD14-transfected cells. Therefore, under physiologic conditions at high SP-A and low LBP concentrations, the ability of SP-A to inhibit immune cell activation by Re-LPS may be due to its ability to block the binding of Re-LPS to LBP and prevent the initiation of the LBP/CD14-dependent pathway for proinflammatory reactions upon Re-LPS stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents/Materials
The Re LPS from Escherichia coli strain F515 was extracted by the phenol/chloroform/petroleum ether method (31), purified, lyophilized, and transformed into the triethylamine salt form. Tritium-labeled LPS from the LCD 25 strain of E. coli K12 (Rb mutant) was obtained from List Biological Laboratories (Campbell, CA). Phosphatidylserine (PS) from bovine brain was from Avanti Polar Lipids (Alabaster, AL) and was used without further purification. Anti-LBP mAb biG 33 IgG1, which is crossreacting with human and rat LBP, and recombinant human (rh) CD14 were obtained from Biometec (Greifswald, Germany). RPMI 1640 medium, Dulbecco's modified Eagle's medium (DMEM), and Dulbecco's phosphate-buffered saline (DPBS) were from GIBCO BRL (Paisley, UK). Poly (dI/dC) was purchased from Pharmacia (Freiburg, Germany). All other chemicals (except as noted) were from Sigma (Deisenhofen, Germany).

Protein Purification
SP-A (generously provided from J. R. Wright, Department of Cell Biology, Duke University Medical Center, Durham, NC) was purified from the bronchoalveolar lavage of patients with alveolar proteinosis (AP) as described in detail (17). SP-A preparations were tested for the presence of bacterial endotoxin using a Limulus Amebocyte Lysate assay (Bio-Whittaker, Walkersville, MD); all SP-A preparations used contained < 0.2 pg endotoxin/µg SP-A. rhLBP was purified by the method of Theofan and coworkers (32) from supernatants of Chinese Hamster Ovary cells transfected with the cDNA of LBP and was a kind gift of S. F. Carroll, XOMA (US) LLC, Berkely, CA.

Isolation of Cells and Incubation Conditions
Alveolar macrophages were isolated by lung lavage of male Sprague-Dawley rats weighing 200–250 g (Charles River, Sulzfeld, Germany) as previously described (21). Cell recovery rouinely averaged 4–7 x 106 cells/animal. The viability of the cells was determined by erythrosin B exclusion and averaged 94–98%. Cells were plated at 0.8 x 106/500 µl in 24-well plates (Costar, Bodenheim, Germany) and allowed to attach for 2 h. The medium was then removed and, for some conditions, Re-LPS (1–100 ng/ml) alone or Re-LPS (1–100 ng/ml) preincubated with SP-A (10 µg/ml) for 2 h at 37°C was used for cell stimulation in the absence or presence (0.2–5%) of heat-inactivated fetal calf serum (FCS) (Bio Whittaker, Verviers, Belgium) for 1 h at 37°C. After stimulation, nuclear extracts were analyzed by a standard electrophoretic mobility shift assay (EMSA). In separate experiments, Re-LPS (10 ng/ml) and SP-A (10 µg/ml) were added to the cells simultaneously, followed by a 1-h incubation. For LBP depletion experiments, the cells were preincubated with anti-LBP mAb (20 µg/ml) for 30 min at 37°C before the addition of Re-LPS preincubated with SP-A (1:10 molar ratio) for 1 h in the presence of 5% serum.

Blood for human mononuclear cell preparation was obtained from normal volunteers. Mononuclear cells were isolated by separation from whole blood, using Ficoll-Paque (Pan Biotech, Aidenbach, Germany) as previously described (33). After washing, the cells were plated at 1 x 106/200 µl in 96-well plates (Costar). Cells were stimulated with Re-LPS (10 ng/ml) alone or Re-LPS (10 ng/ml) preincubated with SP-A at a molar ratio ranging from 1:10 to 1:0.01 in the absence or presence of 5% heat-inactivated FCS for 4 h at 37°C, and cell-free supernatants were analyzed for tumor necrosis factor (TNF)-{alpha} concentration. In separate experiments, cells were incubated with rhLBP (0.0022–22 µg/ml) for 2 h before the addition of Re-LPS (10 ng/ml) preincubated with SP-A at a 1:10 molar ratio, and supernatants were assayed for TNF-{alpha} released after 4 h of incubation.

TNF-{alpha} Enzyme-Linked Immunosorbent Assay
TNF-{alpha} concentrations in cell-free supernatants were determined by sandwich enzyme-linked immunosorbent assay (ELISA) as previously described (33). Serial dilutions of human recombinant TNF-{alpha} (Intex AG, Muttenz, Switzerland) provided a standard curve. The results were expressed as pg TNF-{alpha} per ml.

Nuclear Protein Extraction and Nuclear Factor-{kappa}B Activation Assay
After exposing cells to the experimental conditions, nuclear extracts were prepared by a standard method with modifications as described (34). The cells were scraped off of the plates, centrifuged, and resuspended in 400 µl of ice-cold buffer A (10 mM Tris, 5 mM MgCl2, 10 mM KCl, 1 mM EGTA, 0.3 M sucrose, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM ß-glycerol phosphate, and 1.5 µl of protease inhibitor cocktail [Complete]; Roche, Mannheim, Germany). After 15 min on ice, 25 µl of 10% Nonidet P40 was added. The solution was vortexed and nuclei were pelleted by centrifugation. The nuclear pellet was resuspended in 30 µl of buffer B (20 mM Tris, 5 mM MgCl2, 320 mM KCl, 0.2 mM EGTA, 1 mM dithiothreitol, 25% glycerol, and the mixture of protease inhibitors mentioned above) and, after 15 min on ice, lysates were cleared by centrifugation at 14,000 x g for 15 min. The supernatant containing nuclear proteins was transferred to new vials and the protein concentration of each extract was determined using bicinchoninic acid reagent (Pierce, Rockford, IL). The activity of nuclear factor (NF)-{kappa}B in the nuclear extracts was determined by a standard EMSA. Briefly, 2 µg of crude nuclear extract was incubated for 20 min in binding buffer containing 50 µg of poly (dI/dC)/ml with 7.5 fmol of a 32P-labeled double stranded oligonucleotide encoding the consensus NF-{kappa}B site 5'-AGCTCAGAGGGGACTTTCCGAGAGAGC-3' (MWG-Biotech, Ebersberg, Germany). Samples were separated by electrophoresis in 5% polyacrylamide gels for 2 h at 150 V, after which gels were analyzed with a PhosphorImager (Molecular Dynamics, Krefeld, Germany). To demonstrate specificity of DNA–protein interaction, competition experiments using 500-fold excess of unlabeled double-stranded DNA oligonucleotides were performed.

Cell Culture and Transfection
The human embryonic kidney cell line HEK293 (CLR-1573) was from the American Type Culture Collection (Rockville, MD). HEK293 cells were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. HEK293 cells were transfected using an electroporator (Eppendorf, Hamburg, Germany) according to the manufacturer's protocol. In brief, 106 cells were resuspended in 400 µl hypo-osmolar electroporation buffer (Eppendorf) supplemented with 10 µg expression vector pCEP4/CD14 (a kind gift of D. Golenbock, University of Massachusetts Medical School, Worcester, MA). After electroporation at 500 V for 40 µs, the cells were washed with PBS and cultured in DMEM 10% FCS. The selection of transfected cells was started 2 d later by adding 400 µg/ml hygromycin B (Calbiochem, San Diego, CA). The expression of CD14 was confirmed by flow cytometry using monoclonal Ab (clone Mem18) against CD14 (a kind gift of V. Horesji, Institute of Molecular Genetics, Prague, Czech Republic).

LPS Uptake
In the present study we used a tritium-labeled Rb-LPS mutant which was shown to bind to SP-A in a dose-dependent manner (21). [3H]Rb-LPS uptake by immune cells was determined as previously described (35). The term "uptake/binding" is used to refer to the association of [3H]Rb-LPS with the cell and is a measure of both membrane-bound and internalized LPS (36). Briefly, microfuge tubes were incubated overnight at 4°C with 1% low-endotoxin BSA in DPBS and then washed two times with water. Membrane CD14-HEK cells and WT-HEK cells were washed two times by centrifugation. Cells (0.5 x 106) were resuspended into 0.5 ml of incubation buffer (DPBS with 0.9 mM CaCl2 and 0.1% BSA) and incubated either in the absence or presence of 5% heat-inactivated FCS with 10 ng/ml [3H]Rb-LPS alone or 10 ng/ml [3H]Rb-LPS preincubated with 10 µg/ml SP-A. After incubation at 37°C for 20 min, the cells were collected by centrifugation at 200 x g for 10 min, washed, transferred to a new tube to minimize nonspecific adsorption of radioactivity to the tube, and washed again two times by centrifugation. The final pellet was resuspended in incubation buffer, and 0.2 ml was transferred to a scintillation vial. Two milliliters of scintillation fluid were added to each vial, and the radioactivity was measured in a 1414 scintillation counter (Wallac, Freiburg, Germany).

Surface Plasmon Resonance–Binding Analysis
A surface plasmon resonance (SPR) technique (37) using a BIAcore 3000 (BIAcore, Uppsala, Sweden) was employed as a binding assay to detect interaction of LBP, Re-LPS, SP-A, and a preincubated mixture of Re-LPS and SP-A with immobilized liposomes made from phosphatidylserine (PS). For the preparation of the liposomes, PS was suspended in 100 mM KCl, 5 mM HEPES, pH 7, mixed thoroughly, and was sonicated for 1 min. Then, the preparation was intermittently cooled (30 min at 4°C) and heated (30 min at 56°C), and finally stored at 4°C for at least 12 h before the measurement. Twenty microliters of a 100-µM suspension of PS liposomes was injected to obtain an immobilized lipid matrix on a biosensor L1 chip (BIAcore). SP-A (100 nM), LBP (100 nM), LPS (100 µM), or a preincubated mixture of SP-A and Re-LPS (100 nM:100 µM) preformed in the presence of 2 mM CaCl2 were added at 20-µl volumes. The running buffer was 100 mM KCl, 5 mM HEPES, pH 7.0. All experiments were performed at 25°C at a flow rate of 10 µl/min.

Statistics
Data were analyzed with the Mann-Whitney U test or Student's t test for unpaired samples. Values were considered significant when P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SP-A Inhibits Re-LPS–induced NF-{kappa}B Activation in Alveolar Macrophages
We found that Re-LPS–induced NF-{kappa}B activation in rat alveolar macrophages required the presence of 0.2% heat-inactivated FCS for Re-LPS concentrations of 1–10 ng/ml (Figure 1A) , a finding consistent with a previous report from Martin and colleagues (27), demonstrating that Re-LPS (Salmonella minnesota Re595) concentrations for up to 10 ng/ml are below the threshold for LBP-independent TNF-{alpha} release by alveolar macrophages.



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  		Figure 1. Effects of SP-A on Re-LPS–induced NF-{kappa}B translocation in alveolar macrophages. Rat alveolar macrophages (0.8 x 106/well) were stimulated with Re-LPS (1–100 ng/ml) (A), Re-LPS (1–100 ng/ml) preincubated with SP-A (10 µg/ml) (1:40–1:0.4 molar ratio) (B), or the simultaneous addition of Re-LPS (10 ng/ml) plus SP-A (10 µg/ml) (C) in the presence of 0.2% heat-inactivated FCS for 1 h at 37°C and assayed for NF-{kappa}B translocation by EMSA as described in MATERIALS AND METHODS. Competition analysis was performed by adding a 500-molar excess of unlabeled specific (SP) oligonucleotide in the DNA-binding reactions. Data illustrated are from single experiments and are representative of three to five experiments.

 
SP-A alone did not induce NF-{kappa}B activation in alveolar macrophages (Figures 1B and 1C). In contrast, SP-A dramatically inhibited the activation of NF-{kappa}B in alveolar macrophages stimulated with Re-LPS. In the presence of 0.2% heat-inactivated FCS, NF-{kappa}B translocation induced by 1–50 ng/ml Re-LPS was inhibited by ~ 80–90% when Re-LPS was preincubated with 10 µg/ml of SP-A before the addition to the cells (1:40–1:0.8 molar ratio; Figure 1B). Under the same incubation conditions, the simultaneous addition of SP-A (10 µg/ml) plus Re-LPS (10 ng/ml) failed to inhibit Re-LPS–induced NF-{kappa}B activation in alveolar macrophages (Figure 1C). The specifity of NF-{kappa}B binding was confirmed in competition experiments; a 500-fold molar excess of unlabeled NF-{kappa}B abolished binding of the nuclear extracts to the labeled NF-{kappa}B–binding site sequence (Figure 1C).

The effects of rhSP-A (kindly provided by W. Steinhilber, Byk Gulden, Konstanz, Germany) on Re-LPS–induced NF-{kappa}B activation were similar to the effects of alveolar proteinosis (AP)-SP-A, though AP–SP-A was slightly more potent than rhSP-A in inhibiting Re-LPS–induced NF-{kappa}B translocation (data not shown).

SP-A Inhibits Re-LPS–Induced TNF-{alpha} Production by Human Mononuclear Cells
In human mononuclear cells, a significant LPS-induced TNF-{alpha} production has been shown to occur in a serum-independent (33, 38, this study) but LBP-dependent manner (33). In the present study, SP-A alone did not affect TNF-{alpha} baseline release (< 15 pg/ml) by mononuclear cells incubated for up to 4 h with SP-A. In contrast, SP-A inhibited TNF-{alpha} release by mononuclear cells stimulated with Re-LPS in a dose-dependent manner. In the absence of serum, TNF-{alpha} release induced by 10 ng/ml Re-LPS was inhibited by 80–95% when Re-LPS was preincubated with SP-A (range of 1:1–1:10 [Re-LPS:SP-A] molar ratio) (Figure 2) . Under the same incubation conditions, the simultaneous addition of SP-A (10 µg/ml) plus Re-LPS (10 ng/ml) failed to decrease Re-LPS–induced TNF-{alpha} release by mononuclear cells (data not shown).



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  		Figure 2. Effects of SP-A on Re-LPS–induced TNF-{alpha} release by human mononuclear cells. Human mononuclear cells (1 x 106/well) were incubated with Re-LPS (10 ng/ml) or Re-LPS (10 ng/ml) preincubated with SP-A (1:10–1: 0.01 molar ratio) for 4 h at 37°C in the absence of serum and assayed for TNF-{alpha} concentrations in cell-free supernatants by ELISA. The data shown are mean ± SE of three to five independent experiments performed in triplicate. *P < 0.05 when compared with Re-LPS–induced TNF-{alpha} production.

 
LBP Counteracts SP-A–Mediated Inhibition of Re-LPS–Induced Immune Cell Activation
Because sCD14 and LBP are serum proteins that have been well characterized for their abilities to affect the responsiveness of human monocytes and macrophages to LPS, the role of these factors in SP-A–mediated inhibition of LPS-induced cell activation was next examined. Human recombinant LBP at 0.22 and 22 µg/ml (1:1 and 100:1 [LBP: Re-LPS] molar ratio) in a dose-dependent manner counteracted the inhibitory effects of SP-A on Re-LPS–induced TNF-{alpha} production (Figure 3) by mononuclear cells. In contrast, the addition of sCD14 had very little effect on SP-A–mediated inhibition of TNF-{alpha} production upon cell stimulation with Re-LPS (data not shown). In the presence of 5% heat-inactivated FCS, the SP-A–mediated inhibition of Re-LPS–induced NF-{kappa}B activation in alveolar macrophages was abolished and could be restored by blocking LBP with monoclonal anti-LBP antibodies (Figure 4) .



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  		Figure 3. Effects of LBP on SP-A–mediated inhibition of Re-LPS–induced TNF-{alpha} production by human mononuclear cells. Cells (1 x 106/well) were incubated with recombinant human (rh) LBP (100:1–0.01:1 [LBP:Re-LPS] molar ratio) for 2 h at 37°C before the addition of Re-LPS (10 ng/ml) preincubated with SP-A in a 1:10 molar ratio and assayed for TNF-{alpha} release by ELISA. Data shown are mean ± SE of three independent experiments performed in triplicate. **P < 0.001 and *P < 0.01 when compared with Re-LPS/SP-A–induced TNF-{alpha} production in the absence of rhLBP.

 


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  		Figure 4. Effects of anti-LBP Ab on SP-A–mediated inhibition of Re-LPS–induced NF-{kappa}B translocation in alveolar macrophages. Cells (0.8 x 106/well) were incubated with 20 µg/ml anti-LBP mAb for 30 min at 37°C before the addition of 10 ng/ml Re-LPS preincubated with SP-A (1:10 molar ratio) and assayed for NF-{kappa}B translocation by EMSA after a 1 h incubation in the presence of 5% FCS. Data illustrated are from a single experiment and are representative of three experiments.

 
SP-A–Enhanced Cellular Association of [3H]Rb-LPS Does Not Depend on the Expression of mCD14 by HEK293 Cells
We have previously shown that SP-A increases the cellular association of [3H]Rb-LPS with isolated rat alveolar macrophages (35), a process thought to contribute to the clearance of LPS by immune cells. In the present study, the ability of wild-type (WT) HEK293 cells (Figure 5A) and mCD14-transfected HEK cells (Figure 5B) to bind [3H]Rb- LPS alone or [3H]Rb-LPS preincubated with SP-A was determined. As expected, there was no significant difference in [3H]Rb-LPS uptake by WT- or mCD14-HEK cells in the absence of serum (258 ± 111 cpm versus 164 ± 44 cpm, n = 5), whereas the presence of 5% heat-inactivated human serum caused a significant increase in the association of [3H]Rb-LPS to mCD14-HEK cells compared with serum-free conditions (1,598 ± 548 cpm versus 164 ± 44 cpm, n = 5, P < 0.05).




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  		Figure 5. Effects of SP-A on [3H]Rb-LPS uptake by WT- and mCD14-transfected HEK cells. (A) WT-HEK cells were incubated with [3H]Rb-LPS (10 ng/ml) or [3H]Rb-LPS (10 ng/ml) preincubated with SP-A (1:10 molar ratio) in the absence or presence of 5% heat-inactivated FCS. Cellular association of [3H]Rb-LPS was measured after 20 min of incubation at 37°C as described in MATERIALS AND METHODS. Data shown are mean ± SE of three to five independent experiments. *P < 0.05 compared with [3H]Rb-LPS alone in the absence of FCS. **P < 0.05 compared with [3H]Rb-LPS alone in the presence of FCS. (B) mCD14-HEK cells were incubated with [3H]Rb-LPS (10 ng/ml) or [3H]Rb-LPS (10 ng/ml) preincubated with SP-A (1:10 molar ratio) in the absence or presence of 5% heat-inactivated FCS. Cellular association of [3H]Rb-LPS was measured after 20 min of incubation at 37°C as described in MATERIALS AND METHODS. Data shown are mean ± SE of three to five independent experiments. *P < 0.01 compared with [3H]Rb-LPS alone in the absence of FCS. **P < 0.05 compared with [3H]Rb-LPS alone in the presence of FCS.

 
In the absence of serum, [3H]Rb-LPS uptake by both WT- (Figure 5A) and mCD14-HEK cells (Figure 5B) was significantly enhanced in the presence of SP-A compared with non–SP-A conditions. The amount of cell-associated radioactivity in the presence of SP-A did not differ between WT- and CD14-HEK cells.

Next we investigated the effect of SP-A on the serum-dependent binding of [3H]Rb-LPS to WT- and mCD14-HEK cells. In the presence of serum, SP-A increased the amount of cell-associated radioactivity compared with non–SP-A conditions in WT-HEK cells (Figure 5A). In contrast, significantly smaller amounts of [3H]Rb-LPS were bound to mCD14-HEK cells in the presence of SP-A than in the absence of the protein (Figure 5B).

SP-A Inhibits Re-LPS Interaction with LBP
SPR experiments were performed to investigate the binding of SP-A, LBP, Re-LPS, or Re-LPS preincubated with SP-A to immobilized PS liposomes (Figure 6) . The increase in response units in all four traces (a, b, c, d) after the first injection of PS liposomes (20 µl, 100 µM, time point 1) indicates binding of the liposomes to the surface of the chip. Additional injections of PS liposomes (20 µl and 40 µl) did not increase the signal, indicating a complete coverage of the surface with PS (data not shown). Injection of LBP (20 µl, 100 nM) (traces b and c, time point 2) resulted in its binding to the PS surface and this could not be reversed by washing with running buffer. Subsequent injection of SP-A (20 µl, 100 nM) (traces a and b, time point 3) led to a binding of SP-A only to the PS- (trace a) and not to the LBP-doped surface (trace b). The increase and decrease of the signal after addition of SP-A (trace b) resulted from a change of the buffer (additional 2 mM Ca2+). The injection of Re-LPS preincubated with SP-A (20 µl, 100 µM:100 nM) (traces c and d, time point 4) resulted in an increase in the signal which was ~ 20 times lower in comparison to the change of the signal after subsequent injection of SP-A and Re-LPS (trace b, time points 3 and 5) and four times lower in comparison to the change after injection of pure SP-A (trace a, time point 3). The final addition of Re-LPS (20 µl, 100 µM) (traces a, b, c, and d, time point 5) resulted in Re-LPS binding in all of the four experiments; however, the amount of bound Re-LPS was significantly different being highest in trace a.



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  		Figure 6. Effects of preincubation of Re-LPS with SP-A on Re-LPS interaction with LBP. Time kinetics of changes in the response units in surface plasmon resonance experiments upon injection of LBP, SP-A, Re-LPS, and Re-LPS preincubated with SP-A. Time point 1, 20 µl PS liposomes (100 µM) in experiments a, b (upper panel), and c, d (lower panel). Time point 2, 20 µl LBP (100 nM) in experiments b and c. Time point 3, 20 µl SP-A (100 nM) in experiments a and b. Time point 4, 20 µl Re-LPS preincubated with SP-A (100 µM:100 nM) in experiments c and d. Time point 5, 20 µl LPS (100 µM) in experiments a, b, c, and d. Flow parameters and binding conditions are outlined in MATERIALS AND METHODS.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we first investigated the impact of Re-LPS/SP-A interaction on immune cell function and found that SP-A dramatically decreases the LBP-dependent activation of immune cells to Re-LPS. Preincubation of Re-LPS with SP-A in a dose-dependent manner inhibited Re-LPS–induced activation of alveolar macrophages and human mononuclear cells in the presence of low serum concentrations. At high serum concentrations, the inhibitory effect of SP-A on Re-LPS–induced activation was abolished. Anti-LBP antibodies and rhLBP were used for depletion and reconstitution of media, their effects on SP-A–mediated inhibition of Re-LPS were assessed, and the resulting data confirmed that LBP was a pivotal serum-derived component responsible for the observed effects. Because LBP-catalyzed binding of low-dose LPS to CD14 is essential for LPS-induced signaling by macrophages and monocytes, we hypothesized that the mechanism responsible for SP-A–mediated inhibition of the activation of immune cells stimulated with Re-LPS is via inhibiting Re-LPS interaction with LBP. Using a BIAcore optical biosensor, binding competition experiments were performed to demonstrate that preincubation of Re-LPS with SP-A almost completely inhibited Re-LPS binding to LBP, suggesting a competitive binding of Re-LPS to SP-A or LBP. The data support an anti-inflammatory effect for SP-A in regulating the pulmonary response to rough LPS under conditions assumed to occur physiologically.

NF-{kappa}B is an important transcription factor for immune and inflammatory responses and plays a central role in the regulation of cytokine production upon LPS stimulation. We found that SP-A alone did not affect NF-{kappa}B activation in rat alveolar macrophages. Another study (39) has shown that SP-A treatment of TLR2-transfected HEK293 cells does not affect the basal NF-{kappa}B activity. In contrast to these results, SP-A has been shown to activate NF-{kappa}B in the human THP-1 monocytic cell line (18, 23). TNF-{alpha} is a pivotal mediator of immune cell responses to LPS and triggers a wide variety of proinflammatory responses. In the present study, SP-A did not stimulate TNF-{alpha} release by human mononuclear cells, a finding which is consistent with those reported for SP-A by several investigators who found no effects of SP-A on the baseline production of TNF-{alpha} by human monocytes (40), human and rat alveolar macrophages (17, 20, 22, 40), and the U937 human macrophage-like cell line (40). However, they appear to contradict the data from other studies (23, 41) that showed that SP-A could stimulate TNF-{alpha} production by human peripheral blood mononuclear cells and by THP-1 cells. Currently, there is no explanation for these conflicting results. Subtle differences in experimental design, cell specifity, SP-A isolation procedure, and prestimulation of cells may account for the different results.

Assuming molecular masses of 630 and 2.5 kD for SP-A and Re-LPS, respectively, SP-A to Re-LPS molar ratios of 40:1–0.8:1 resulted in a significant inhibition of Re-LPS–induced NF-{kappa}B activation in alveolar macrophages at serum concentrations estimated to occur in the normal air spaces. SP-A is an abundant protein in the human alveolar lining fluid, with a concentration of 200–500 µg/ml as estimated from determinations in diluted bronchoalveolar fluid (42). The concentration of LBP is assumed to be much lower, i.e., 10–100 ng/ml in the undiluted alveolar fluid, corresponding to a serum concentration of ~ 0.1–1% (43). In patients suffering from acute lung injury, bronchoalveolar concentrations of LBP have been documented to rise by ~ 100-fold (30), whereas SP-A concentrations significantly decrease (42). The affinity of the Re-LPS/SP-A interaction is not known. LBP binds to the lipid A moiety of R- and S-LPS with a dissociation constant near 10-9 M (6). Our observation that the simultaneous addition of SP-A plus Re-LPS fails to inhibit Re-LPS–induced immune cell activation in the presence of LBP corroborates the assumption that the binding affinity of LBP for Re-LPS is higher than that of SP-A for Re-LPS. In addition, we found that the inhibitory effects of SP-A on Re-LPS–induced cellular activation was completely abolished when the preincubation of SP-A with Re-LPS was performed in the presence of serum (data not shown), suggesting that a serum component with higher affinity for LPS than SP-A directly competes with SP-A for LPS binding, e.g., formation of a complex. Together the data suggest that, under physiologic conditions, the large excess of SP-A in the pulmonary compartment in the presence of low LBP and low LPS concentrations may play a major role in regulating the sensitivity of immune cells to Re-LPS by preventing the initiation of the LBP/CD14 pathway for inflammatory reactions.

We have previously shown that SP-A increases the cellular association of tritium-labeled Rb-LPS with isolated rat alveolar macrophages (35) independent of pretreatment of the cells with phosphatidylinositol-specific phospholipase C, which removes mCD14 from the outer cell membrane. In the present study, we extend those observations by employing mCD14 transfection studies on HEK293 cells, demonstrating that the SP-A–enhanced cellular association of [3H]Rb-LPS under serum-free conditions was independent of the expression of mCD14. Because the amount of cell-associated radioactivity in the presence of SP-A did not differ between WT- and mCD14-HEK cells, an alternative, mCD14-independent pathway of SP-A–mediated LPS cell association might be assumed. In addition, SP-A did promote the LPS uptake in the presence of serum by WT-HEK cells, but inhibited the serum-enhanced interaction of [3H]Rb-LPS with mCD14-HEK cells, suggesting that the preincubation of [3H]Rb-LPS with SP-A interferes with the serum-mediated binding of [3H]Rb-LPS to mCD14.

In addition to the mechanism of SP-A–mediated inhibition of LPS-induced activation proposed in this study, direct modulation of immune cells via interaction of SP-A with cellular binding sites is likely to be involved in immunomodulatory effects of SP-A. Sano and coworkers (20) showed that SP-A directly interacts with CD14. The authors proposed that the effects of SP-A on both S- and R-LPS–induced TNF-{alpha} occur via direct interaction of SP-A with CD14 (20). In their study, SP-A inhibited S-LPS–induced TNF-{alpha} release, but enhanced TNF-{alpha} production by rat alveolar macrophages upon stimulation with LPS from Re595-Salmonella minnesota in the presence of 10% serum. By preincubation of soluble sCD14 with SP-A, the binding of sCD14 to R-LPS was significantly increased, whereas the binding of sCD14 to S-LPS was decreased. The observed differences in binding reported by Sano and colleagues (20) and the present study may be related to the ability of the different assays used, different structural equirements of CD14 for the binding to different forms of LPS, the presence of soluble accessory molecules (LBP), and the form of CD14 (soluble versus membrane-bound). Importantly, is has been shown that the soluble and membrane forms of CD14 have different structural determinants for LPS receptor function (44).

In the present study, we found that SP-A significantly inhibits Re-LPS–induced NF-{kappa}B translocation in rat alveolar macrophages in the presence of 0.2% serum. In addition, the SP-A–mediated inhibition of Re-LPS–induced cellular activation was dependent on a preincubation step of SP-A with Re-LPS, suggesting that the direct interaction of SP-A with Re-LPS has a significant impact on the cell response to Re-LPS. In fact, SPR experiments revealed that the interaction of Re-LPS with SP-A significantly interferes with the subsequent binding of Re-LPS to LBP. We thus propose that the inhibitory effect of SP-A on Re-LPS–induced cell activation is due to an inhibited or reduced transfer of LPS to CD14 by LBP. However, the combined data suggest that SP-A can inhibit different phenotypes of LPS by distinct mechanisms. The SP-A–mediated inhibition of Re-LPS–induced activation observed in our study may largely depend on the local LBP concentration and/or the concentration of other serum-derived LPS-binding proteins such as amyloid A and P, albumin, and low-density lipoprotein (45, 46).

The expression pattern of SP-A shows predominant localization in the lung, where SP-A has been shown to interact with alveolar macrophages in a specific and high-affinity manner (47). In addition, there is increasing evidence for extrapulmonary production of SP-A (48, 49), findings of which the physiologic relevance remains to be established. In contrast to alveolar macrophages (27, this study), human mononuclear cells respond to LPS at pico- and nanomolar concentrations by TNF-{alpha} production in a serum-independent manner (33, 38, this study). The LPS-induced activation of mononuclear cells was found to be inhibited by monoclonal antibodies to LBP (33), suggesting the existence of membrane-associated LBP for these cells. In the present study, SP-A significantly inhibited Re-LPS–induced TNF-{alpha} production by mononuclear cells under serum-free conditions, and increasing concentrations of LBP or serum (unpublished data) abrogated the SP-A–mediated inhibition of Re-LPS–induced TNF-{alpha} release. These data confirm the crucial role of LBP in modulating the SP-A–mediated neutralization of Re-LPS.

In conclusion, this study demonstrates that SP-A inhibits the LBP-dependent activation of immune cells by Re-LPS. We propose that the effect of SP-A may be due to its ability to block the binding of Re-LPS to LBP and prevent the initiation of the LBP/CD14 pathway for inflammatory responses to Re-LPS. Future studies will investigate whether SP-A modifies Re-LPS-induced activity in the lung in vivo under physiologic and pathophysiologic conditions.


   Acknowledgments
 
The authors thank Katrin Klopfenstein, Sabrina Groth, and Christine Hamann for excellent technical assistance. They are grateful to Jo Rae Wright who supplied the human SP-A preparations, and to Stephen F. Carroll for the recombinant LBP. They are also indebted to all volunteers who donated blood for this study. This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) SFB 367/B8 (to U.S.) and DFG 609/1-1 (to C.S.).

Received in original form January 3, 2002

Received in final form April 25, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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