Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo

Paul Borron1, J. Clarke McIntosh2, Thomas R. Korfhagen3, Jeffrey A. Whitsett3, Julie Taylor1, and Jo Rae Wright1

Departments of 1 Cell Biology and 2 Pediatrics, Duke University, Durham, North Carolina 27710; and 3 Division of Pulmonary Biology, Children's Hospital Research Foundation, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of surfactant-associated protein (SP) A in the mediation of pulmonary responses to bacterial lipopolysaccharide (LPS) was assessed in vivo with SP-A gene-targeted [SP-deficient; SP-A(-/-)] and wild-type [SP-A(+/+)] mice. Concentrations of tumor necrosis factor (TNF)-alpha , macrophage inflammatory protein-2, and nitric oxide were determined in recovered bronchoalveolar lavage fluid after intratracheal administration of LPS. SP-A(-/-) mice produced significantly more TNF-alpha and nitric oxide than SP-A(+/+) mice after LPS treatment. Intratracheal administration of human SP-A (1 mg/kg) to SP-A(-/-) mice restored regulation of TNF-alpha , macrophage inflammatory protein-2, and nitric oxide production to that of SP-A(+/+) mice. Other markers of lung injury including bronchoalveolar fluid protein, phospholipid content, and neutrophil numbers were not influenced by SP-A. Data from experiments designed to test possible mechanisms of SP-A-mediated suppression suggest that neither binding of LPS by SP-A nor enhanced LPS clearance are the primary means of inhibition. Our data and others suggest that SP-A acts directly on immune cells to suppress LPS-induced inflammation. These results demonstrate that endogenous or exogenous SP-A inhibits pulmonary LPS-induced cytokine and nitric oxide production in vivo.

lipopolysaccharide; lung inflammation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PULMONARY SURFACTANT is a mixture of phospholipids, neutral lipids, and proteins that coats the alveolar surface, the site of gas exchange. This mixture reduces the surface tension at the air-liquid interface of the alveoli, preventing collapse at end expiration (20, 21). Surfactant-associated protein (SP) A is an abundant phospholipid-associated protein that plays a role in the formation of tubular myelin and in host defense. SP-A is a member of a family of calcium-dependent lectins (collectins) that includes serum mannose binding protein, SP-D, and conglutinin (29, 38, 44). Collectins, including SP-A, have a collagen-like amino-terminal domain and a globular COOH-terminal carbohydrate recognition domain that binds various carbohydrates including D-mannose, L-fucose, D-galactose, and D-glucose (1, 2, 16, 46). SP-A binds to a variety of bacterial and viral pathogens as well as to lipopolysaccharide (LPS) from some serotypes of bacteria (10).

Collectins have multiple host defense functions. For example, collectins bind to immune cells, opsonize and enhance bacterial clearance, and affect oxygen radical production (36, 37, 39, 40, 49-52, 54). In addition, SP-A acts as an anti-inflammatory agent in vitro, suppressing LPS-induced production of tumor necrosis factor (TNF)-alpha by alveolar macrophages and inhibiting mitogen-induced T-cell proliferation and interleukin (IL)-2 production in vitro (6, 31). Contradictory results to these findings have been reported with different culture systems and methods of SP-A purification (23, 25).

Recent studies (17, 22, 26, 27) with SP-A-deficient [SP-A(-/-)] mice showed that the absence of SP-A has a minimal effect on surfactant homeostasis but a major impact on bacterial clearance. LeVine et al. (26) showed that clearance of intratracheal group B streptococcus and Pseudomonas aeruginosa from the lungs of SP-A(-/-) mice was impaired compared with clearance from the lungs of wild-type [SP-A(+/+)] mice. Decreased clearance of bacteria was associated with decreased binding and uptake by alveolar macrophages. Systemic spread of group B streptococcus infection was also increased in SP-A(-/-) mice (26, 27). Intratracheal administration of exogenous human SP-A restored bacterial killing in SP-A(-/-) mice at 6.67 and 4.44 mg/kg but not at 2.22 mg/kg (27). Concentrations of TNF-alpha and macrophage inflammatory protein-2 (MIP-2; the murine homolog for IL-8) (8) in lung homogenates were increased in SP-A(-/-) mice compared with those in SP-A(+/+) mice after infection with P. aeruginosa (26).

These studies showed that the bacterially infected SP-A(-/-) mice had higher levels of cytokines and chemokines than the SP-A(+/+) mice. However, it was unclear whether the increases were a direct effect of SP-A on pulmonary cytokine production or related to the decreased capacity of SP-A(-/-) mice to clear bacteria. In the present study, the role of SP-A in the regulation of inflammatory responses to LPS was assessed in vivo. Inflammatory cytokines, nitric oxide (NO) production, and other markers of lung injury were measured in SP-A(+/+) mice, SP-A(-/-) mice, and SP-A(-/-) mice treated with exogenous SP-A after intratracheal administration of LPS.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Animal care and treatment. The gene encoding murine SP-A was disrupted by homologous recombination to generate an inbred strain of SP-A-deficient mice. It was previously shown that neither SP-A mRNA nor protein can be detected in these mice (22). Mice of either sex, 20-24 wk of age, and ranging in weight from ~25 to 32 g were housed under pathogen-free conditions until they were used for experiments. SP-A(+/+) mice were of the same genetic background (129J) and were age matched. The animals were anesthetized by exposure to gauze soaked in Metaphane (methoxyflurane; Mallinckrodt Veterinary) in a closed jar until the mice were no longer conscious and breathing had slowed. Mice were then restrained on a Plexiglas board and continuously exposed to Metaphane from a soaked gauze pad placed inside a 15-ml conical tube. After the skin was swabbed with 70% ethanol, an incision of ~1.5 cm in length was made 2 cm above the sternum to expose the trachea. A 27-gauge needle attached to a 1-ml syringe (Becton Dickinson) containing 100 µl of either 70 µg/kg (2 µg/animal) of Escherichia coli LPS 026:B6 (Sigma) or 70 µg/kg of LPS and 1 mg/kg (30 µg/animal) of human SP-A was inserted into the trachea. The fluid was injected slowly, and the incision was closed with 35 R Reflex One (Richard-Allen) surgical staples.

Animals were killed 3 h after injection. Previous studies (2, 18, 33, 48, 56) in rats and mice demonstrated that peak concentrations of TNF-alpha were present after intratracheal administration of LPS doses ranging from 10 ng/kg to 0.5 mg/kg 3-6 h after instillation. Animals were given an intraperitoneal injection of 200 µl of Nembutal (pentobarbital sodium; Abbott Laboratories). After loss of both consciousness and the ability to respond to tail or foot pad squeezes, the surgical staples were removed, the trachea was cannulated, and the mice were killed by exsanguination. The lungs were exposed by removal of the front portion of the rib cage and lavaged three times with 0.8 ml of lavage buffer. Lavage buffer consisted of phosphate-buffered saline-2 mM EDTA (pH 7.4) prepared with endotoxin-free water (Picopure Water, Hydro Water Management Systems, Research Triangle Park, NC). BAL fluid was chilled on ice in 15-ml conical tubes before centrifugation at 400 g for 10 min. The volume of cell-free lavage fluid recovered was measured for each animal. The samples were stored in aliquots in sterile 1.5-ml microfuge tubes at -80°C. The number of mice and method of experimentation were approved by the Duke University (Durham, NC) Medical Center Institutional Animal Care and Use Committee.

Isolation of human SP-A. SP-A was isolated as previously reported (55). BAL fluid from patients undergoing therapeutic lavage for treatment of alveolar proteinosis was obtained with approval of the Duke University Medical Center Institutional Review Board. The large aggregates of surfactant were allowed to settle for at least 24 h under unit gravity. Removal of lipid and hydrophobic proteins from the pellet was achieved by butanol extraction with subsequent high-speed centrifugation of the butanol-lavage mixture (105 g for 60 min). On evaporation of butanol, the insoluble pellet was resuspended in 100 mM octylglucopyranoside, 150 mM NaCl, and 5 mM Tris, pH 7.4, and mixed with polymyxin-agarose (1:5 vol/vol; Sigma) for 30 min at room temperature. To remove octylglucopyranoside, the mixture was dialyzed (14,000 mol wt cutoff) for a minimum of four complete changes of buffered endotoxin-free water (5 mM Tris, pH 7.4) followed by another high-speed centrifugation to remove insoluble proteins. The supernatant was then collected and stored at -20°C. Protein concentration was determined with a micro-bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) and bovine serum albumin as a standard. Identity of the isolated protein was confirmed by SDS-PAGE and Western blot analysis with a rabbit polyclonal anti-human SP-A antiserum as previously reported (32). The SP-A used for the study was assayed for endotoxin content, which was found to be 0.013 pg endotoxin/µg protein.

Analysis of cytokines. Both the L929 bioassay (30) and ELISA were used to determine TNF-alpha concentrations in BAL fluid so that a comparison between immunoreactive TNF-alpha and bioactive TNF-alpha could be made. For the bioassay, serial dilutions of lavage sample (37.5 µl/well) were tested in duplicate alongside buffer controls containing no TNF-alpha . These samples were added to L929 cells (American Type Culture Collection, Manassas, VA) that had been plated in 96-well plates at a concentration of 3 × 105 cells in 75 µl tissue culture medium/well. MEM (with glutamine and Earle's salts; GIBCO BRL) tissue culture medium was supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% horse serum (GIBCO BRL). After 24 h in culture, the medium was replaced with serum-free MEM with 0.2% bovine serum albumin (fraction V, low endotoxin; Sigma), actinomycin D (2 µg/ml; Sigma), and the diluted lavage sample. L929 cells were incubated for a further 24 h with sample, standard, or buffer before being fixed with 5% formaldehyde for 5 min, stained with 1% crystal violet for 5 min, and dried. TNF-alpha concentrations were calculated by comparing the decrease in the absorbance at 540 nm to decreases obtained with serial dilutions of recombinant mouse TNF-alpha (R&D Systems).

ELISA for inflammatory cytokines. TNF-alpha or MIP-2 concentrations in BAL fluid were determined with assay kits from Genzyme Diagnostics and R&D Systems, respectively, in conjunction with a Bio-Rad model 550 microplate reader with accompanying software (Bio-Rad, Hercules, CA) as directed by the manufacturer. BAL samples were diluted 1:10 and 1:25 in the diluent provided with the assay. TNF-alpha and MIP-2 standards were individually added to BAL samples to ensure that BAL fluid constituents did not interfere with the assay. The range of detection for the TNF-alpha and MIP-2 ELISA kits were 35-2,240 and 7.8-500 pg/ml, respectively.

Determination of nitrite recovered from lung lavage. Nitrite, which is a by-product of NO production, was assayed in the BAL fluid with the method of Schmidt et al. (45). Griess reagent (1 mM sulfanilamide, 1 mM napthylethylenediamine, and 0.1 M HCl) was added to each sample (1:1 vol/vol) in a 96-well tissue culture plate and incubated for 10 min at room temperature. The color change in the samples was quantified with a spectrophotometric plate reader (540 nm) and compared with dilutions of a known standard (NaNO2).

Cellular content of BAL fluid. Cells from BAL samples were isolated by centrifugation at 400 g for 10 min. The cells were resuspended in 1 ml of RPMI 1640 tissue culture medium and 10% (by volume) fetal bovine serum (GIBCO BRL). The cells were diluted 1:1 with Turk's solution and counted with a hemocytometer. Approximately 1-2 × 105 cells from each sample were used to prepare cytospins in a Cytospin 2 (Shandon, PA) centrifuge at a speed of 450 rpm for 5 min. These slides were then air-dried, stained with the Hemacolor stain set (EM Diagnostic Systems), and permanently mounted with a glass coverslip. A minimum of 200 cells/sample were counted and evaluated.

BAL fluid lipid and protein. The method of Bligh and Dyer (5) was used to extract lipids from the mouse lavage fluid before assay of these extracted samples for inorganic phosphorus by the method of Bartlett (1). Protein concentrations were determined with a micro-BCA protein assay kit and bovine serum albumin as a standard.

Measurement of LPS-SP-A interactions. FITC-labeled E. coli LPS 026:B6 (20 µg) was added to 300 µg of SP-A in Dulbecco's PBS (GIBCO BRL) plus 1 mM Ca2+ and Mg2+ (total volume 1 ml) and incubated for 3 h at 37°C in the dark. The LPS-SP-A sample was dialyzed for 24 h (in the dark) at room temperature against three 1-liter changes of Dulbecco's PBS (plus Ca2+ and Mg2+) with Slide-A-Lyzer dialysis cassettes (mol wt cutoff of 10,000). Simultaneously, a FITC-LPS control (no protein) was used in the same procedure. The resulting dialysates were analyzed for FITC-LPS with a spectrofluorimeter. The amounts of FITC-LPS contained in the control (no SP-A included) and experimental samples were calculated by generating a standard curve of relative fluorescent units versus known amounts of FITC-LPS. The FITC-LPS used as a standard was obtained from the same stock solution added to the binding assay. Furthermore, the SP-A used for the binding assay was from the same preparation of SP-A used for in vivo experiments. The protein content of the SP-A-LPS sample was measured with the micro-BCA assay kit after the 24 h of dialysis.

Statistics. Statistical analysis was performed with the Primer for Biostatistics computer program and manual (13). Analysis of variance was used to determine differences among experimental groups. A multiple comparison procedure, the Student-Newman-Keuls test, was used. A P value of <= 0.05 was considered to be significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SP-A inhibits TNF-alpha after LPS treatment. Significantly more bioactive (Fig. 1) and immunoreactive (Fig. 2) TNF-alpha was recovered in the BAL fluid from SP-A(-/-) mice compared with that from SP-A(+/+) mice 3 h after intratracheal administration of 70 µg/kg of LPS. Coadministration of human SP-A and LPS to SP-A(-/-) mice decreased TNF-alpha concentrations in BAL fluid to 84% of values measured in SP-A(+/+) mice (control). Amounts of immunoreactive TNF-alpha in the BAL fluid from untreated SP-A(+/+) or SP-A(-/-) mice were similar and were approximately fivefold lower than those of LPS-treated SP-A(+/+) mice.


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Fig. 1.   Surfactant-associated protein (SP) A inhibits production of bioactive tumor necrosis factor (TNF)-alpha in vivo. A TNF-alpha bioassay with L929 cells was performed on mouse bronchoalveolar lavage (BAL) fluid obtained 3 h after intratracheal injection of lipopolysaccharide (LPS; 70 µg/kg) into wild-type (+/+) mice, SP-A-deficient (-/-) mice, and SP-A(-/-) mice treated with LPS and human SP-A (1 mg/kg; rescue). Data are percent of concentration in BAL fluid from SP-A(+/+) mice (control); n = 6 mice/group. * P <=  0.05 compared with wild-type control mice.



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Fig. 2.   SP-A inhibits production of immunoreactive TNF-alpha in vivo. A TNF-alpha ELISA was performed on mouse BAL fluid obtained 3 h after intratracheal (it) administration of LPS into SP-A(+/+) (n =17), SP-A(-/-) (n =16), and rescue (n = 15) mice. Hatched bars represent TNF-alpha content in BAL fluid of mice that were not challenged with LPS. Amount of TNF-alpha in BAL fluid from SP-A(+/+) mice averaged 26.1 × 103 ± 2.9 × 103 pg TNF-alpha /lung. * P <=  0.05 compared with SP-A(-/-) mice. ** P <=  0.05 compared with SP-A(+/+) mice.

SP-A inhibits MIP-2 after LPS challenge. MIP-2 in BAL fluid increased after LPS treatment (Fig. 3) in both SP-A(-/-) and SP-A(+/+) mice. Coadministration of SP-A and LPS to SP-A(-/-) mice decreased MIP-2 concentrations in BAL fluid below amounts detected in either SP-A(-/-) or SP-A(+/+) mice challenged with LPS. MIP-2 was not detectable in the BAL fluid from SP-A(+/+) or SP-A(-/-) mice that did not receive LPS (data not shown).


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Fig. 3.   LPS-stimulated macrophage inflammatory protein (MIP)-2 production is suppressed by SP-A. MIP-2 was analyzed by ELISA with the same lavage samples assayed for TNF-alpha in Fig. 2. BAL fluid was obtained 3 h after intratracheal injection of LPS into SP-A(+/+) (n = 17), SP-A(-/-) (n = 16), or rescue (n = 15) mice. Amount of MIP-2 in BAL fluid from SP-A(+/+) mice averaged 10.8 × 103 ± 1.7 × 103 pg/lung (n = 17). ** P <=  0.05 compared with SP-A(-/-) mice.

SP-A inhibits BAL fluid nitrite after LPS treatment. NO production was analyzed in the BAL fluid from mice from all three experimental groups and control animals by quantifying nitrite, a product created when NO reacts with O2 in solution (12). The amount of nitrite was greater in BAL fluid from SP-A(-/-) mice compared with that from SP-A(+/+) mice. Coadministration of LPS and human SP-A rescued the SP-A(-/-) phenotype by significantly reducing the amount of nitrite in the BAL fluid from SP-A(-/-) mice compared with amounts detected in the BAL fluid from SP-A(-/-) mice (Fig. 4). There was detectable nitrite in the BAL fluid from untreated SP-A(+/+) (n = 3) or SP-A(-/-) (n = 3) mice that was 80.3 ± 33.7 and 122 ± 60.4%, respectively, of that from LPS-treated SP-A(+/+) mice.


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Fig. 4.   Nitrite concentration in BAL fluid after LPS challenge is decreased by exogenous SP-A. Griess reaction was used to analyze nitrite concentration in BAL samples obtained 3 h after intratracheal injection of LPS into SP-A(+/+) (n = 17), SP-A(-/-) (n = 16), and rescue (n = 15) mice. Average concentration of nitrite in SP-A(+/+) mice was 1.66 ± 0.57 µmol/lung. * P <=  0.05 compared with SP-A(+/+) mice. ** P <=  0.05 compared with SP-A(-/-) mice.

SP-A did not influence other indexes of pulmonary inflammation. LPS treatment increased BAL fluid protein in both SP-A(-/-) and SP-A(+/+) mice. No significant differences were detected in the concentration of protein or phospholipid recovered from LPS-treated SP-A(-/-) or SP-A(+/+) mice (Table 1). Coadministration of SP-A and LPS did not alter the concentration of BAL fluid phospholipid or protein from SP-A(-/-) mice compared with that from SP-A(+/+) mice (Table 1).

                              
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Table 1.   Total protein and phospholipid recovered by BAL from LPS-treated SP-A(+/+) and SP-A(-/-) mice

LPS treatment increased the number and altered the types of cells in the BAL fluid from SP-A(+/+) and SP-A(-/-) mice (Table 2). Greater than 90% of the BAL fluid cells were alveolar macrophages in untreated animals. The percentage of macrophages dropped to ~50% and neutrophils increased to ~50% in all three of the LPS treatment groups (Table 3).

                              
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Table 2.   Percentage of alveolar macrophages and neutrophils obtained by BAL from LPS-treated mice


                              
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Table 3.   Cell counts in BAL fluid after LPS treatment

SP-A binds minimal amounts of E. coli LPS 026:B6 in solution. After 3 h of incubation at 37°C, only 5.8 ng FITC-labeled LPS 026:B6/µg SP-A were bound. We calculated that the maximum amount of LPS 026:B6 that could have bound SP-A after 3 h in vitro was 8.65%.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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Summary. We have shown here that SP-A-deficient mice are more sensitive to intratracheal endotoxin than wild-type mice. For example, production of biologically active and immunoreactive TNF-alpha in the airways of SP-A(-/-) mice was greater than that in SP-A(+/+) mice challenged with intratracheal LPS. In addition, the amount of nitrite measured in the BAL fluid from SP-A(-/-) mice was greater than the amount in SP-A(+/+) mice. Cotreatment of SP-A(-/-) mice with human SP-A and LPS rescued the knockout phenotype. Specifically, the amounts of TNF-alpha , MIP-2, and nitrite were similar in SP-A(+/+) mice and SP-A(-/-) mice treated with SP-A. However, the amounts of protein and phospholipid and the number of infiltrating neutrophils did not vary among the three experimental groups tested. Thus SP-A appears to act as an anti-inflammatory protein because it inhibits LPS-induced cytokine and NO production in vivo. However, SP-A does not totally protect the lung from LPS-induced inflammation in the experimental model tested.

Effects of SP-A on cytokine and nitrite production. Inhibition of cytokine production by SP-A has been reported by several groups. For example, McIntosh et al. (31) previously reported that SP-A inhibits LPS-induced (E. coli 026:B6 and 0111:B4) TNF-alpha production by alveolar macrophages and that SP-A treatment of a mixed culture of lung fibroblasts and LPS-activated alveolar macrophages protected fibroblast growth. It was also found that SP-A inhibits IL-2 production by human peripheral blood mononuclear cells stimulated in vitro with mitogen (7). Similarly, Sano et al. (43) reproduced the finding that SP-A inhibits LPS-induced TNF-alpha production by a macrophage-like tumor cell line stimulated with different serotypes of LPS. Recently, Cheng et al. (9) showed that SP-A decreased ionomycin-induced IL-8 production and release by human eosinophils. Hickling et al. (14) demonstrated that SP-A reduced production of TNF-alpha by buffy coat cells stimulated with P. aeruginosa LPS. Rosseau et al. (41) examined the effect of SP-A on cytokine production by monocytes and macrophages treated with Candida albicans and showed that SP-A inhibited TNF-alpha production by both cell types. Further analysis of cytokine production by alveolar macrophages showed that the amount of several proinflammatory mediators (IL-1, IL-8, MIP-1 and monocyte chemoattractant protein-1) was reduced by SP-A, and it was suggested that SP-A acted directly on monocytes and macrophages to suppress cytokine production (41). These in vitro reports are consistent with our conclusion that SP-A exerts an anti-inflammatory effect in vivo, perhaps by several different mechanisms.

Our results also show that SP-A reduces the amount of nitrite in lavage fluid from LPS-challenged mice. These results are consistent with those from a study by LeVine et al. (26), which demonstrated that levels of nitrite were greater in SP-A(-/-) mice than in SP-A(+/+) mice after intratracheal infection with P. aeruginosa.

In contrast to our results and the studies summarized above, SP-A has been shown to stimulate cytokine and NO production (4, 23, 25). For example, Kremlev and Phelps (23) reported that SP-A stimulates the production of TNF-alpha , IL-1alpha , IL-1beta , and IL-6 by rat splenocytes. SP-A also enhanced the production of immunoglobulins A, G, and M by rat splenocytes as well as their proliferation (23, 24). Blau et al. (4) reported that SP-A, LPS, and combinations of the two stimulated production of nitrite by alveolar macrophages.

There are several possible explanations for these conflicting observations. For example, SP-A is purified by several different methods, and its activity may be dependent on this variable (6, 7). The means by which cells are activated to elaborate cytokines is also important. The in vitro studies have investigated responses of a variety of cell types including cultured splenocytes and differentiated THP-1 cells, and it is possible that SP-A may exert cell-specific effects. In addition, the effects of SP-A appear to vary with the type of pathogen stimulus. For example, Hickman-Davis et al. (15) demonstrated that SP-A increased the nitrite production of alveolar macrophages stimulated with Mycoplasma pneumoniae. In contrast, Pasula et al. (35) reported that SP-A suppresses reactive nitrogen intermediates by murine alveolar macrophages exposed to Mycobacterium tuberculosis. In any case, our studies show that endogenous SP-A in the alveolar milieu can suppress some proinflammatory responses that are induced by LPS.

Not all indexes of inflammation were altered by SP-A. Exogenous or endogenous SP-A did not alter the amount of LPS-induced protein leak, phospholipid, or cellular profile in BAL fluid. These results suggest that the decreases in the detectable amounts of cytokines were not an artifactual result of differences in total recoverable protein. Our results are consistent with the finding that pretreatment of rats with dexamethasone before LPS challenge decreased TNF-alpha accumulation in BAL fluid but not the total amount of protein in BAL fluid (28, 34). Inhibition of some but not all indexes of inflammation suggests that that SP-A has selective actions in vivo or that its action as an anti-inflammatory compound is not potent enough to attenuate more potentially sensitive markers of lung injury.

There was no significant difference in the total amount of MIP-2 recovered from the LPS-challenged lungs of SP-A(+/+) or SP-A(-/-) mice despite the fact that TNF-alpha concentrations were significantly different in the same samples. Data from the referenced study by Xing et al. (56) showed that LPS-induced MIP-2 and TNF-alpha mRNA expression in the whole rat lung as well as in alveolar macrophages differed in several ways. For example, the amount of LPS-induced MIP-2 mRNA appeared greater than TNF-alpha mRNA in the whole lung and alveolar macrophages at the earliest tested time points (30 min and 1 h, respectively). MIP-2 mRNA expression was also much higher than TNF-alpha at much later time points (12 and 24 h, respectively). These data suggest that MIP-2 production, like protein leak and cellular influx, is a more sensitive index of inflammation and one on which SP-A does not exert an effect unless present in high concentrations.

Two lines of evidence suggest that SP-A can inhibit MIP-2 production under certain circumstances. LeVine et al. (26) showed that MIP-2 production was greater in lung tissue homogenates from SP-A(-/-) mice than in those from SP-A(+/+) mice after infection with P. aeruginosa. Our results with exogenous human SP-A (rescue group) show that coadministration of SP-A and LPS to SP-A(-/-) mice does decrease the amount of MIP-2 measured in BAL fluid relative to that in BAL fluid from SP-A(-/-) mice or SP-A(+/+) mice challenged with LPS.

We do not know definitively why exogenously administered SP-A was a more effective inhibitor of MIP-2 production than endogenous SP-A. One explanation is that when the exogenous SP-A was administered concurrently with LPS to ensure codistribution of LPS and SP-A, an acute inflammatory response occurred in the context of a larger than normal concentration of SP-A. It is also possible that exogenous SP-A exerts a more potent inhibitory response before associating with lipid. However, it has been previously shown (31) that the anti-inflammatory effect of SP-A on LPS (026:B6)-stimulated alveolar macrophages was maintained in the presence of surfactant-like lipids and that the magnitude of this inhibition was comparable to SP-A added in the absence of lipid. It is important to note that the dose of exogenous SP-A administered to SP-A(-/-) mice is estimated to be within a physiological range. For example, Ryan et al. (42) estimated that the lungs from rats weighing 250-300 g contain 600-720 µg recoverable SP-A/kg body weight.

Possible mechanisms by which SP-A inhibits LPS-stimulated inflammation. SP-A may inhibit LPS-induced cytokine production in vivo by several different mechanisms. It is possible that SP-A blocks LPS interactions with immune cells, possibly by inhibiting the interaction of LPS with cells by binding CD14 (43) or conversely binding to LPS and preventing its interaction with the cell surface (19). In vitro data also raise the possibility that SP-A increases the clearance of LPS from the lung (47).

To address the possibility that SP-A-mediated decreases in cytokine production exclusively through LPS binding and clearance from the airways, SP-A(+/+) and SP-A(-/-) mice and a rescue group received an intratracheal injection of FITC-LPS (E. coli 026:B6, 2 µg/animal). Animals were lavaged 1 h after instillation rather than 3 h due to limitations of detecting fluorescence above background. The amounts of fluorescence detected in the cell-free lavage fluid and the 150-g pellet were similar in the three groups [SP-A(+/+), SP-A(-/-), and rescue; 7 animals/group]. These findings are particularly important because it has been shown that optimum LPS-induced stimulation of cultured monocytes occurs in a matter of minutes (11). These data suggest that SP-A is not exclusively exerting its anti-inflammatory effect through enhanced LPS clearance.

Blau et al. (3) and Kalina et al. (19) have previously reported that interactions of SP-A with LPS can affect their functions. For example, they showed that conditioned medium from SP-A-treated alveolar macrophages stimulates the in vitro formation of myeloid progenitor cell colonies to a similar magnitude as conditioned medium from LPS- or IL-1-treated alveolar macrophages (3). A subsequent report found that coculturing SP-A and LPS with alveolar macrophages reduced the colony-forming activity in the medium compared with macrophages treated with SP-A or LPS alone. The observed mechanism for the inhibition of both LPS and SP-A stimulatory activity was correlated with LPS-SP-A binding (E. coli strain 346) (19).

For our study, we used a serotype of LPS that was reported not bind to SP-A. This serotype of LPS was chosen to minimize the possibility that the mechanism of immune suppression of SP-A is through the binding of LPS. A previous study (43) showed that SP-A does not bind to E. coli LPS 026:B6. These results were confirmed in another binding assay in which SP-A did not bind LPS 026:B6 but did bind E. coli LPS LCD25 (C. Stamme and J. R. Wright, unpublished observations). Additionally, we designed a binding assay to determine whether SP-A bound LPS 026:B6 in solution because adherence of LPS to a microtiter plate may alter its binding properties. The soluble binding assay showed that when 100 µg of SP-A were incubated with 20 µg of LPS 026:B6, it bound only 0.00577 µg LPS/µg SP-A after a 3-h incubation (total SP-A bound 8.65% of total LPS). Although we cannot exclude the possibility that this very low amount of LPS binding is sufficient to inhibit LPS-stimulated cytokine production, these data as well as the previous finding that SP-A can suppress TNF-alpha production by alveolar macrophages when SP-A is added several hours after LPS activation (31) suggest that SP-A inhibition of LPS-induced inflammation is by means of a mechanism that does not require SP-A to physically interact with LPS.

Physiological relevance of SP-A with respect to the pulmonary immune system. Use of SP-A-deficient mice has established that SP-A functions in the resolution of pulmonary bacterial infections and regulation of the resulting pulmonary inflammation. These findings are consistent with other in vitro observations that SP-A inhibits a variety of potentially proinflammatory responses including T-lymphocyte proliferation, IL-2 production (7, 53), and IL-8 production (9) as well as nitrite production (35). The in vivo data presented here and the growing number of reports confirming our initial in vitro observation that SP-A also acts as an anti-inflammatory molecule suggest that a fundamental role of SP-A within the airways is immunologic homeostasis.


    ACKNOWLEDGEMENTS

This research was supported by National Heart, Lung, and Blood Institute Grants HL-51134 (to J. R. Wright), HL-58795 (to T. Korfhagen and J. R. Wright), and HL-28623 (to J. A. Whitsett); by a Clinical Research Grant from the March of Dimes (to J. C. McIntosh); and by fellowships from the Canadian Lung Association and the Parker B. Francis Foundation (to P. Borron).


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. R. Wright, Dept. of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710 (E-mail: j.wright{at}cellbio.duke.edu).

Received 23 March 1999; accepted in final form 8 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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