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Published ahead of print on May 28, 2003, doi:10.1164/rccm.200212-1440OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 335-341, (2003)
© 2003 American Thoracic Society

Cellular Antiendotoxin Activities of Lung Surfactant Protein C in Lipid Vesicles

Luis A. Augusto, Monique Synguelakis, Quentin Espinassous, Michel Lepoivre, Jan Johansson and Richard Chaby

Endotoxin Group, and Laboratory of Nitrogen Oxides Inflammation and Immunity, UMR-8619, National Center for Scientific Research, University of Paris-Sud, Orsay, France; and Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Correspondence and requests for reprints should be addressed to Richard Chaby, M.D., Equipe Endotoxines, UMR-8619 du CNRS, Bâtiment 430, Université de Paris-Sud, 91405 Orsay, France. E-mail: richard.chaby{at}bbmpc.u-psud.fr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The respiratory system is continuously exposed to airborne particles containing lipopolysaccharide. Our laboratory established previously that the hydrophobic surfactant protein C (SP-C) binds to lipopolysaccharide and to one of its cellular receptors, CD14. Here we examined the influence of SP-C, and of a synthetic analog, on some cellular in vitro effects of lipopolysaccharide. When associated with vesicles of dipalmitoylphosphatidylcholine, SP-C inhibits the binding of a tritium-labeled lipopolysaccharide to the macrophage cell line RAW 264.7. Under similar conditions of presentation, SP-C inhibits the mitogenic effect of lipopolysaccharide on mouse splenocytes, and inhibits the lipopolysaccharide-induced production of tumor necrosis factor-{alpha} by peritoneal and alveolar macrophages, and of nitric oxide by RAW 264.7 cells. In contrast, tumor necrosis factor-{alpha} production induced by a lipopeptide, and nitric oxide production induced by picolinic acid, were not affected by SP-C. The lipopolysaccharide-binding capacity of SP-C is resistant to peroxynitrite, a known mediator of acute lung injury formed by reaction of nitric oxide with superoxide anions. These results indicate that SP-C may play a role in lung defense; SP-C resists degradation under inflammatory conditions and traps lipopolysaccharide, preventing it from inducing production of noxious mediators in alveolar cells.

Key Words: lipopolysaccharide • nitric oxide • peroxynitrite • surfactant protein-C • tumor necrosis factor-{alpha}

Bacterial lipopolysaccharide (LPS) is a component of the gram-negative bacterial cell wall that is released in vivo into the circulation during infection and sepsis. A common clinical complication of sepsis is lung injury, with frequent development of an acute respiratory distress syndrome (1). LPS may also be shed from the outer bacterial membrane into the environment, where it is ubiquitously present. Airborne LPS has been detected in several occupational environments associated with conditions such as swine worker's disease (2), cotton dust worker's disease (3), farmer's disease (4), and lung disease in the fiberglass industry (5). The presence of LPS in house dust (6, 7), and its possible role in domestic asthma (8, 9), have also been reported. Because of inhalation of airborne particles containing bacteria and LPS, the respiratory system is continuously exposed to this potent proinflammatory compound. In normal subjects, acute inhalation of LPS induces systemic (10) and airway inflammation (11), in the absence of significant change in lung function (12). However, long-term exposure to LPS, as occurs in agricultural workers, results in a profound inflammatory response and is associated with increased risk of developing lung diseases, including chronic obstructive pulmonary disease. These pulmonary effects of LPS show the importance of understanding how inflammatory reactions are initiated, perpetuated, or inhibited in the lung.

The lung has efficient defense mechanisms against LPS under normal exposure conditions. When LPS carried by gram-negative bacteria or dust reaches the alveolar spaces at the terminal airways, it encounters a surfactant layer that covers the epithelium, and consists of lipids and surfactant-associated proteins. It has been established that the two hydrophilic surfactant proteins (SP)-A and SP-D are important in pulmonary host defense, not only because they bind and aggregate various microorganisms (13), but also because they bind LPS and modulate LPS-triggered responses (14, 15). The two hydrophobic surfactant proteins SP-B and SP-C are considered critical for the absorption and spreading of the surfactant film at the air–liquid interface, but their possible role in lung defense remains elusive. We established, however, in previous studies that SP-C, but not SP-B, can bind to the lipid A region of LPSs of various phenotypes (16, 17). We also found (18) that SP-C interacts with CD14, a coreceptor for LPS expressed by macrophages and other cell types (19). The aim of this study was to determine whether these molecular interactions influence the responses of immunocompetent cells to LPS.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media, Reagents, and Cells
Culture medium (CM) was RPMI 1640 (GIBCO, Grand Island, NY) containing 2 mM L-glutamine, penicillin (100 IU/ml), and streptomycin (100 µg/ml). Dipalmitoylphosphatidylcholine (DPPC) and picolinic acid were from Sigma (St. Louis, MO). The mitogenic tripalmitoyl pentapeptide (MTPP) was from Bachem (Bubendorf, Switzerland). Tritium-labeled thymidine (925 GBq/mmol), from Amersham-Pharmacia Biotech (Buckinghamshire, UK), was diluted to 150 GBq/mmol with unlabeled thymidine before use.

The two rough-type LPSs from Salmonella enterica serovar minnesota, chemotypes Re (strain Re-595) and Rc (strain R5), as well as the rough-type LPSs from Salmonella enterica serovar typhimurium (rough chemotype Rc, strain SL 1181) and Escherichia coli (chemotype Rd2, strain F583), and the smooth-type LPS from Klebsiella pneumoniae, were from Sigma. The LPS from the Re-595 strain of Salmonella enterica serovar minnesota was tritium labeled ([3H]LPS) by a modification of the procedure of Watson and Riblet (20) as described previously (17).

Porcine SP-C was isolated by organic extraction of minced lung tissue followed by Lipidex 5000 and Sephadex LH-60 chromatography (21). Mouse crude surfactant was isolated from the bronchoalveolar lavage (BAL) fluid of 5- to 10-week-old Swiss mice (R. Janvier, Le Genest Saint-Isle, France), on an NaCl–NaBr density gradient, as described by Katyal and coworkers (22), and mouse SP-C was purified from this material by reversed-phase high-performance liquid chromatography as described previously (16). SP-C/BR and SP-C(LKS) were synthesized by t-Boc chemistry and isolated by reversed-phase high-performance liquid chromatography as described (23–25). For experiments involving incubation with cells, dried mixtures (sterilized under ultraviolet light) of purified surfactant components (2 µg) and DPPC (18 µg) were sonicated in 1 ml of CM, and 100 µl of the vesicles obtained was added to the cells in a final volume of 250 µl. It has been established that the vesicles obtained by this technique are multilamellar, and heterogeneous in size (80–500 nm), with a phase transition temperature of 40.1°C (26). The phospholipid:protein ratio of these vesicles (90:10 by weight) is similar to that of natural surfactant, and our previous study (16) indicated that their apparent density on a salt gradient is also similar to that of natural surfactant. The binding of tritium-labeled LPS to SP-C was analyzed as described previously (16), with lipid vesicles loaded with SP-C and containing bovine serum albumin (added to reduce nonspecific binding). It has been shown (27) that the presence of bovine serum albumin in the vesicles has little impact on their size and density.

Peroxynitrite was synthesized from reaction of gaseous nitric oxide with hydrogen peroxide as described previously (28). Concentration in peroxynitrite was measured using the molar extinction coefficient of 1,670 M–1 cm–1 at 302 nm.

The mouse monocyte-macrophage cell line RAW 264.7 (European Collection of Cell Cultures [ECCAC, Salisbury, UK] catalog number 91062702) was maintained as an adherent culture in CM supplemented with 10% heat-inactivated and endotoxin-free fetal calf serum (FCS).

Alveolar and peritoneal macrophages were collected from normal, uninfected Swiss outbred mice (6 to 12 weeks of age). Alveolar macrophages were harvested from mouse lungs by bronchoalveolar lavage. Peritoneal exudates were harvested 5 days after intraperitoneal injection of 1.7 ml of thioglycollate broth. Alveolar and peritoneal cells were centrifuged, resuspended in CM, and immediately plated in 24-well plastic plates. After 3 hours at 37°C in a 5% CO2 atmosphere, nonadherent cells were removed by two or three washings with RPMI 1640.

Cellular Assays
The binding of tritium-labeled LPS to macrophages was analyzed as described previously (29). Tumor necrosis factor-{alpha} (TNF-{alpha}) present in culture supernatants 24 hours after stimulation was measured by specific sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (eBioscience, San Diego, CA). Nitric oxide production was estimated by measuring the nitrite concentration of the cell supernatants by the Griess reaction (30). Analysis of the mitogenic effect of LPS was performed on mouse spleen cells (2.5 x 105 cells per well). The cells were incubated for 60 hours (37°C, 5% CO2) in CM supplemented with 5% FCS and 10-5 M 2-mercaptoethanol, in the presence of LPS (0.2 to 1 µg/ml) preincubated with DPPC vesicles (400 µg/ml) with or without the synthetic peptide SP-C(LKS) (400 pmol/ml). [3H]thymidine (150 GBq/mmol; 22 kBq/well) was then added and the cells were reincubated for 8 hours. The incorporated radioactivity was collected on glass fiber filters in a cell harvester (Skatron, Lier, Norway), and counted.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of LPS Binding to RAW Cells, and LPS-induced Production of TNF-{alpha}
We demonstrated in previous studies that native SP-C of human or animal origin, and several synthetic analogs of these molecules, interact with the lipid A moiety of LPSs (17). Because macrophages play a central role in the pathophysiological effects of LPS, we analyzed the influence of SP-C on the binding of LPS to this cell type, and on the resulting cellular effects. We first used the mouse monocyte-macrophage cell line RAW 264.7, which is considered a good model of LPS-binding and LPS response (31, 32). In an initial experiment, we examined the influence of various SP-C peptides on the production of TNF-{alpha} induced by LPS in this cell line. We compared porcine SP-C, mouse SP-C, and SP-C (LKS), which is a nonpalmitoylated synthetic analog of SP-C. The structures of these molecules are represented in Figure 1 . Because SP-C and its synthetic analogs are hydrophobic and almost insoluble in aqueous culture media, these peptides were incorporated into vesicles of DPPC as described previously (16). Suspensions of vesicles with or without natural or synthetic SP-C were mixed with the rough-type LPS Re-595 (SP-C:LPS molar ratio of 25, corresponding to mouse lungs infected with half a billion bacteria), and incubated for 48 hours at 37°C with RAW cells. The experiment was performed in the absence of serum. The results (Figure 2) show that a marked inhibition of LPS-induced production of TNF-{alpha} was observed in the presence of vesicles containing porcine or mouse SP-C, or SP-C(LKS) (94, 55, and 67% inhibition, respectively), as compared with the response induced in the presence of void vesicles. Because the effect of mouse SP-C was not significantly different from that of SP-C(LKS), we used the latter in all further experiments. The influence of SP-C(LKS) (400 pmol/ml) on the production of TNF-{alpha} induced by different LPSs (40 ng/ml) in RAW cells was also examined. We found that there was no statistically significant differences between the inhibitory effects of SP-C(LKS) on the TNF-{alpha} production induced by five different LPSs, including the smooth-type LPS from K. pneumoniae, and four rough-type LPSs from Salmonella and E. coli (data not shown).



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  		Figure 1. Structures of natural and synthetic analogs of SP-C used. Palm = palmitoyl residues.

 


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  		Figure 2. Influence of various types of SP-C on the LPS-induced production of TNF-{alpha} by RAW cells. Monolayers of RAW 264.7 cells (5 x 105 cells per well) were washed and exposed (48 hours at 37°C in CM without serum) to LPS from S. minnesota Re-595 (40 ng/ml) in the presence of vesicles of DPPC (200 µg/well) alone, or containing 200 pmol/well of natural SP-C or its synthetic analog SP-C(LKS). Results are expressed as the amount of TNF-{alpha} secreted by 106 cells, and represent means ± SD of three determinations.

 
The inhibition of cytokine production in the presence of SP-C can be due either to a molecular neutralization of LPS by SP-C, or to a cellular effect of SP-C. In view of our previous demonstration of a direct interaction between LPS and SP-C (16, 17), the former scenario is the most likely, and implies that SP-C may influence the binding of LPS to its cellular receptors. Therefore, we analyzed the influence of SP-C(LKS) on the binding of tritium-labeled LPS to RAW cells. The cells were incubated for 1 hour at 0°C with [3H]LPS, either with SP-C(LKS) in suspension or with SP-C(LKS) in lipid vesicles. Because serum constituents such as LPS-binding protein (LBP) or high-density lipoproteins (HDLs) can influence LPS binding to cells (33, 34), the experiment was performed either in the presence or in the absence of serum. The results (Figure 3A) show that SP-C(LKS) carried by lipid vesicles induced a marked inhibition of LPS binding, whereas no effect was observed with a suspension of the synthetic peptide. This is likely because SP-C, which is highly hydrophobic, leads to aggregates in aqueous media, in which the LPS-binding site of the peptide is much less accessible. We also observed that the presence of serum in the binding medium did not influence these results. In a second experiment (Figure 3B), [3H]LPS was mixed and preincubated for 2 hours with suspended or vesicle-bound SP-C(LKS) before exposure to the cells. In this experiment, a partial inhibition of LPS binding was observed with the suspension of the peptide. However, the effect was much higher with the peptide in lipid vesicles and, again, the presence of serum appeared irrelevant. Taken together, the results in Figures 2 and 3 show that the interaction between LPS and SP-C(LKS) markedly reduces the capacity of LPS to bind to cells and to induce production of TNF-{alpha}. Because optimal conditions for this effect are obtained with the peptide loaded on lipid vesicles, and do not require serum, we used these conditions in all further experiments.



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  		Figure 3. Influence of SP-C(LKS) on the binding of [3H]LPS to RAW cells. [3H]LPS (3.6 x 105 cpm) was mixed with CM or vesicles of DPPC (200 µg/well), without (solid columns) or with 200 pmol/well of SP-C(LKS) (open columns). The mixtures (250 µl) were added immediately (A), or after 2 hours of preincubation at 20°C (B), to RAW 264.7 cells (106 cells per well) in CM (250 µl), without serum or with 5% FCS. After 1 hour at 0°C, and three washings with 0.15 M NaCl at 0°C, the cell-bound radioactivity was recovered with 0.5 ml of 10% SDS. Data represent means ± SD of three determinations.

 
Inhibition of LPS-induced Production of TNF-{alpha} in Alveolar and Peritoneal Macrophages
To ensure that the results described above are not restricted to the cell line used, we analyzed the influence of SP-C(LKS) on LPS-induced TNF-{alpha} in mouse alveolar and peritoneal macrophages. Similar effects were observed. With alveolar macrophages (Figure 4) , lipid vesicles containing SP-C(LKS) (200 pmol/well) induced a 68% inhibition of the TNF-{alpha} secretion in response to LPS at 40 ng/ml (8 pmol/well). With thioglycollate-elicited peritoneal macrophages (Figure 5A) , an 80% inhibition of the TNF-{alpha} response was obtained with an SP-C(LKS):LPS molar ratio of 25 (synthetic peptide at 200 pmol/well, and LPS at 40 ng/ml). At lower SP-C(LKS):LPS ratios (LPS concentrations higher than 50 ng/ml), the ability of SP-C(LKS) to neutralize the LPS effect decreased progressively.



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  		Figure 4. Influence of SP-C(LKS) on the LPS-induced production of TNF-{alpha} by alveolar macrophages. Mouse alveolar macrophages (3.5 x 105 cells per well) were exposed (48 hours at 37°C in CM without serum) to various concentrations of LPS from S. minnesota Re-595, in the presence of vesicles of DPPC (200 µg/well) alone (solid circles), or containing 200 pmol/well of the synthetic peptide SP-C(LKS) (open circles). Results are expressed as the amount of TNF-{alpha} secreted by 106 cells, and represent means ± SD of three determinations.

 


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  		Figure 5. Influence of SP-C(LKS) on the production of TNF-{alpha} by peritoneal macrophages stimulated with LPS or MTPP. Mouse peritoneal macrophages (106 cells per well) were exposed (24 hours at 37°C in CM without serum) to various concentrations of LPS from S. minnesota Re-595 (A), or to MTPP (100 ng/ml) (B) in the presence of vesicles of DPPC (200 µg/well) alone (solid symbols) or containing 200 pmol/well of the synthetic peptide SP-C(LKS) (open symbols). Results are expressed as the amount of TNF-{alpha} secreted by 106 cells, and represent means ± SD of three determinations.

 
To ensure that the inhibitory effect of SP-C(LKS) was not due to some general inhibition of TNF-{alpha} synthesis, we used as a TNF-{alpha} inducer a lipopeptide structurally unrelated to LPS: the mitogenic tripalmitoyl pentapeptide (MTPP). We found (Figure 5B) that at 100 ng/ml, this agent induced a low but highly significant production of TNF-{alpha}. However, unlike that of LPS, this effect was not inhibited in the presence of SP-C(LKS). Therefore, this result shows that SP-C(LKS) does not block the ability of macrophages to produce TNF-{alpha}.

Inhibition of LPS-induced Production of Nitric Oxide in RAW Cells
Another important response involved in the antimicrobial activities of macrophages is the production of nitric oxide. Macrophage nitric oxide synthase is the product of a transcriptionally inducible (iNOS) gene, whose maximal expression requires IFN-{gamma} plus a second stimulus (35). In particular, the RAW 264.7 monocyte-macrophage cell line can be efficiently activated by LPS and IFN-{gamma} to produce NO (35, 36). We thus analyzed the capacity of SP-C(LKS) to block this effect. Like TNF-{alpha}, a 77% inhibition of the production of nitric oxide was obtained with an SP-C(LKS):LPS molar ratio of 25 (synthetic peptide at 200 pmol/well, and LPS at 40 ng/ml) (Figure 6) . Again, at lower SP-C(LKS):LPS ratios (LPS concentrations higher than 40 ng/ml), the ability of SP-C(LKS) to neutralize the LPS effect decreased progressively. The inhibitory effect of SP-C(LKS) is dose dependent (Figure 7A) , and maximal at an SP-C(LKS):LPS molar ratio of 25 (Figures 6 and 7A).



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  		Figure 6. Inhibition of LPS-induced nitric oxide production by SP-C(LKS) in RAW cells. Monolayers of RAW 264.7 cells (5 x 105 cells per well) were washed and exposed (48 hours at 37°C in CM without serum) to various concentrations of LPS from S. minnesota Re-595, in the presence of IFN-{gamma} (5 U/ml) and of vesicles of DPPC (200 µg/well) alone (solid circles), or containing 200 pmol/well of the synthetic peptide SP-C(LKS) (open circles). Nitrite values used to estimate NO production represent the mean ± SD of six determinations with samples consisting of three replicates of culture supernatants harvested from two distinct macrophage cultures.

 


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  		Figure 7. Influence of SP-C(LKS) on the production of nitric oxide by RAW cells stimulated with LPS or picolinic acid. Monolayers of RAW 264.7 cells (5 x 105 cells per well) were washed and exposed (48 hours at 37°C in CM without serum) to a 40-ng/ml concentration of LPS from S. minnesota Re-595 (A), or to picolinic acid (B), in the presence of IFN-{gamma} (5 U/ml), and of vesicles of DPPC (200 µg/well) containing various amounts of the synthetic peptide SP-C(LKS). Nitrite values used to estimate NO production represent the mean ± SD of six determinations with samples consisting of three replicates of culture supernatants harvested from two distinct macrophage cultures.

 
To check the specificity of this inhibitory effect, we used picolinic acid, an end catabolite of L-tryptophan that has been reported to act as a costimulus with IFN-{gamma} for the induction of NO production (37). We confirmed that picolinic acid stimulates NO production in RAW cells. However, SP-C(LKS) did not reduce this effect significantly, even when used at relatively high concentrations (800 pmol/well) (Figure 7B). Therefore, SP-C(LKS) does not block the activation of the iNOS gene, and suggests that as for TNF-{alpha}, the inhibition of LPS-induced NO production by SP-C(LKS) is likely due to a direct neutralization of LPS.

Peroxynitrite Does Not Influence the Interaction between LPS and SP-C
Nitric oxide produced by macrophages can react with superoxide anion produced by the same cells, to form peroxynitrite, a strong oxidant that causes lipid peroxidation (38), and oxidation of specific amino acid residues of proteins such as cysteine, methionine, tyrosine, and tryptophan (39). It has been shown that peroxynitrite is produced in the lung during acute endotoxemia (40). Therefore, it was conceivable that peroxynitrite could oxidize SP-C, and thus destroy its capacity to interact with LPS, which would lead to an amplification of the endotoxin effects. The peptide SP-C/BR (see structure in Figure 1) is appropriate for testing this putative damaging mechanism because this synthetic analog of SP-C binds efficiently to LPS (17), and because it contains four possible oxidation sites: two cysteine residues in its hydrophilic N-terminal region (residues 5 and 6), and two tyrosine residues in its hydrophobic C-terminal region (residues 28 and 35). This peptide and mouse SP-C were exposed to peroxynitrite under conditions used previously (28): dried aliquots of mouse SP-C (176 pmol) and of SP-C/BR (200 pmol) were sonicated in 40 µl of a phosphate buffer containing diethylene triamine pentaacetic acid (100 µM). A solution (5 µl) of peroxynitrite (200 µM) was then added. After incubation for 5 minutes at 20°C under constant sonication, the suspensions were lyophilized. The dried materials were incorporated into DPPC vesicles, which were tested for their capacity to bind tritium-labeled LPS. The maximal LPS-binding index (74 ± 3%) was reached with about 200 pmol of mouse SP-C and SP-C/BR. The results in Table 1 show that treatment with peroxynitrite did not significantly modify the ability of natural SP-C or SP-C/BR to bind LPS, as compared with samples treated with the buffer alone. When dissolved in a chloroform–methanol (1:1 by volume) mixture containing 0.14 M triethylamine, the peroxynitrite-treated sample of SP-C/BR exhibited an absorption maximum at 415 nm similar to that of 3-nitrotyrosine, corresponding to 0.35 nitro residue per mole of SP-C/BR.


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  		TABLE 1. Influence of peroxynitrite treatment on the lipopolysaccharide-binding capacity of surfactant protein c

 
Inhibition of the Mitogenic Activity of LPS in Mouse Spleen Cells
B lymphocytes are present in the lung (41) and in that location may play an important role in tumor surveillance (42). LPS stimulates B lymphocytes by inducing the proliferation and polyclonal activation of these cells. We thus examined whether SP-C(LKS) can influence the mitogenic activity of LPS. This was measured on spleen cells. The results in Figure 8 show that the LPS-induced incorporation of [3H]thymidine in mouse spleen cells is partially inhibited by the presence of SP-C(LKS). Therefore, as with macrophages, SP-C(LKS) blocks the response of spleen cells to LPS.



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  		Figure 8. Influence of SP-C(LKS) on the mitogenic effect of LPS. Mouse spleen cells (2.5 x 105 cells per well) were incubated for 60 hours (37°C, 5% CO2) in CM supplemented with 5% FCS and 10-5 M 2-mercaptoethanol, with various concentrations of LPS from S. minnesota Re-595, in the presence of vesicles of DPPC (400 µg/ml) alone (solid circles), or containing synthetic peptide SP-C(LKS) (400 pmol/ml) (open circles). After additional incubation (8 hours) with [3H]thymidine (150 GBq/mmol; 22 kBq/well), incorporated radioactivity was measured. Data represent means ± SD of three determinations.

 
Evaluation of the Antiendotoxin Activity of SP-C in the Blood Circulation
Among various strategies for the control of endotoxin-mediated pathophysiological disorders, early therapy targeting the neutralization of LPS is probably the best approach. Therefore, the ability of SP-C to block cellular effects of LPS in vitro led us to ask whether this peptide can be used in vivo as a therapeutic agent to reduce endotoxin effects. Four mice were injected intravenously, either with LPS from S. minnesota Re-595 alone (25 ng in 0.1 ml saline), or with the same amount of LPS bound to SP-C(LKS)–DPPC vesicles. Ten animals were also injected with SP-C(LKS)–DPPC vesicles without LPS. After 1 hour, the animals were bled, and individual plasma levels of TNF-{alpha} were determined. The results (Table 2) show that the plasma levels of TNF-{alpha} induced by LPS in the presence or absence of SP-C(LKS) were not significantly different (p = 0.5, Student t test). This suggests that the LPS-neutralizing activity of SP-C is lost in the blood environment.


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  		TABLE 2. Influence of sp-c(lks) on plasma levels of tumor necrosis factor-{alpha} induced by intravenous injection of lipopolysaccharide

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We established previously (16–18) that SP-C, and analogs thereof, can bind to bacterial LPS. This feature is common to a large panel of proteins, such as the polymyxin B of gram-positive bacteria, the cecropin of insects, the anti-LPS factor of the arthropod Limulus polyphemus (LALF), the magainin of amphibian skin, and a number of mammalian peptides such as lactoferrin, hemoglobin, and high-density lipoprotein (HDL) in blood, the acute-phase LPS-binding protein (LBP), the bactericidal permeability-increasing protein (BPI) of neutrophil granules, and a heparin-binding protein (also called CAP-37). However, not all proteins with LPS-binding capacity have the same influence on the physiological effects of LPS. For example, hemoglobin, LBP, and CAP-37 enhance LPS-induced cytokine production (43–45), whereas others (BPI and HDL) block these effects (46, 47). These opposite effects are used to optimize the response to LPS during bacterial infection by upregulation of the beneficial LPS signal that alerts the host to the presence of invading pathogens, and downregulation of the detrimental LPS responses that trigger inappropriate and excessive inflammation.

It was therefore necessary to determine which of these upregulating or downregulating effects was promoted by SP-C. For this purpose we used a rough-type LPS to ensure that the observed effect is mediated by lipid A, the biologically active region of endotoxin, and also because several bacteria infecting the airways (Haemophilus influenzae, Bordetella pertussis, and Neisseria meningitidis) have LPSs with short carbohydrate chains (lipooligosaccharides) (48–50). We first examined the influence of SP-C on the binding of LPS to a macrophage cell line. SP-C in lipid vesicles clearly inhibited the binding, but was considerably less active in suspension without lipids (Figure 3). This is understandable inasmuch as SP-C is probably the most hydrophobic protein yet encountered in mammals, and can form insoluble aggregates (51, 52) that may not favor proper interactions with LPS. In contrast, the presence of serum had no influence on the SP-C-induced inhibition of the binding of LPS to macrophages. Therefore, in all further experiments, cells were exposed to lipid-associated SP-C in the absence of serum, to avoid the influence of other components with LPS-binding capacity, such as LBP or HDL.

An important mediator of the pathophysiological effects of LPS is the secretion of TNF-{alpha} by macrophages. We found that SP-C blocks this LPS-induced response in peritoneal and alveolar macrophages, and in a macrophage cell line. Therefore, SP-C functions like HDL and BPI, which both block the cytokine responses of macrophages stimulated by LPS.

Our results (Figures 5 and 6) indicated that optimal inhibition is obtained when the molecular SP-C:LPS ratio is equal to 25, or higher. Because each bacterial cell contains about 106 molecules of LPS, and because mouse lungs contain about 5 x 1014 molecules of SP-C (3.6 µg), this means that during infection, SP-C in mouse lungs can accommodate a maximum of half a billion (0.5 x 109) bacteria, which is a physiologically relevant situation.

A specific molecule does not necessarily have the same effect on all cellular responses to LPS. For example, LBP, which enhances in macrophages the production of TNF-{alpha} (44), has no influence on the production of nitric oxide by this cell type (53). We thus analyzed the effect of SP-C on NO production in RAW 264.7 cells, and found that this LPS-induced production is also blocked by SP-C. This inhibition of NO production may be of critical importance because it should lead to an inhibition of peroxynitrite, which is formed by reaction of NO with superoxide anions, and plays a major role in acute lung injury and acute respiratory distress syndrome (54). If a degradation of the LPS-binding site of SP-C by this reactive agent had been possible, it would lead to a loop of sustained or autoamplified inflammation. This is apparently not the case, and we found that the LPS-binding site of SP-C seems insensitive to peroxynitrite, unlike the activity of whole surfactant and SP-A, reported to be damaged by peroxynitrite (55, 56).

The inhibitory activity of SP-C on macrophage responses to LPS can be due either to its capacity to neutralize LPS, or to interference with a biochemical event in the signaling cascade, leading to activation of the TNF-{alpha} and iNOS genes. The latter possibility can be excluded because we found that SP-C has no influence on the levels of TNF-{alpha} induced by MTPP, and on the production of NO triggered by picolinic acid. Therefore, it seems reasonable to suppose that SP-C acts by direct and specific neutralization of LPS, and this hypothesis is supported by the observation that SP-C also blocks the mitogenic effect of LPS on splenocytes, and the binding of LPS to RAW 264.7 cells.

We established that SP-C also binds to CD14, and enhances the binding of LPS to CD14 in solution or expressed by granulocytes (18). However, in the present study, we found that SP-C did not enhance but rather inhibited the binding of LPS to RAW 264.7 cells, although these cells express normal levels of membrane CD14. This discrepancy probably reflects the fact that in macrophages other membrane constituents such as scavenger receptors, TLR4, and CD11/CD18 may contribute much more than CD14 to LPS binding (57, 58).

The inhibiting effect observed in this study indicates that the surfactant component SP-C probably plays a major role in lung defense mechanisms by trapping LPS and preventing it from triggering its inflammatory effect on alveolar cells. The efficiency of SP-C, as compared with other surfactant components, could be due in part to its high abundance (65% of the surfactant proteins on a molar basis), and in part to the high resistance of its LPS-binding site to degradation by peroxynitrite. When artificially introduced in vivo in tissue other than lung tissue, the LPS-neutralizing activity of SP-C can be blunted by the presence of other constituents. This is particularly the case in circulating blood, where the synthetic analog of SP-C cannot block LPS-induced production of TNF-{alpha} in vivo. This is likely due to a well known phenomenon: the redistribution of lipids and hydrophobic molecules in the circulation (59, 60). SP-C, like other hydrophobic drugs, can be redistributed from liposome vesicles to circulating lipoproteins, or to lipid transfer protein, and thus become unavailable for neutralization of LPS. Lipoproteins and lipid transfer protein, which are abundant in the blood, are normally not present in the alveolar space, and this may allow lung SP-C to interact with LPS under physiological conditions. Further investigations are required to confirm this hypothesis.


   FOOTNOTES
 
Supported by grants from the Direction des Systèmes de Forces et de la Prospective (contract 99.34.033) and the Swedish Research Council.

Received in original form December 10, 2002; accepted in final form May 15, 2003


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