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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 373-378, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0071OC

Surfactant Protein B Inhibits Endotoxin-Induced Lung Inflammation

Ralph Epaud, Machiko Ikegami, Jeffrey A. Whitsett, Alan H. Jobe, Timothy E. Weaver and Henry T. Akinbi

Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Address correspondence to: Henry T. Akinbi, M.D., Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail: henry.akinbi{at}chmcc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transgenic mice, in which the level of surfactant protein (SP)-B mature peptide varied 5.6-fold between SP-B(+/-) and SP-B–overexpressing lines (SP-B+/+/+), were used to test the hypothesis that SP-B protects against endotoxin-induced lung inflammation. Intratracheal administration of endotoxin resulted in significantly lower concentration of SP-B mature peptide and elevated levels of total protein in bronchoalveolar lavage fluid of SP-B(+/-) mice compared with SP-B–overexpressing mice, indicating that endotoxin treatment leads to impairment of SP-B expression coincident with increased lung injury in SP-B(+/-) mice. Recruitment of inflammatory cells and elaboration of proinflammatory cytokines in bronchoalveolar lavage fluid were reduced in SP-B–overexpressing mice compared with SP-B(+/-) mice, suggesting that SP-B inhibited endotoxin-induced lung inflammation. Lung compliance and tissue damping were significantly decreased in SP-B(+/+) and SP-B(+/-) mice, but were not changed in SP-B(+/+/+) mice, consistent with a protective effect of SP-B. The minimum surface tension of large aggregate surfactant was significantly lower for surfactant isolated from SP-B–overexpressing mice, both in the absence and the presence of added plasma proteins. These data suggest that SP-B protected against endotoxin-induced lung inflammation by enhancing surfactant function, resulting in reduced lung injury, decreased influx of inflammatory cells, and lower cytokine levels; in contrast, levels of SP-B in SP-B(+/-) mice were further decreased by endotoxin treatment, likely exacerbating lung injury in this group.

Abbreviations: bronchoalveolar lavage fluid, BALF • enzyme-linked immunosorbent assay, ELISA • interleukin, IL • phosphate-buffered saline, PBS • surfactant protein, SP • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Alveolar stability at low lung volumes is maintained by the formation and maintenance of a surfactant film at the air–liquid interface of the alveolus. A key component of the surface film is dipalmitoylphosphatidylcholine, which reduces surface tension to low levels at end–expiration, thereby preventing alveolar collapse. The formation and stability of the surfactant phospholipid film is highly dependent upon the presence of specific hydrophobic peptides associated with surfactant phospholipids, including surfactant protein (SP)-B and SP-C. Mutations in the human SP-B gene that lead to the complete absence of SP-B protein are also associated with aberrant processing of the SP-C proprotein and decreased levels of SP-C mature peptide in the alveolar airspaces (1). SP-B deficiency results in neonatal respiratory distress syndrome that is generally fatal despite intensive postnatal respiratory support (2–4). A similar outcome is observed in SP-B(-/-) mice, which fail to inflate their lungs and die from respiratory failure following birth (5, 6).

In addition to postnatal lethality caused by loss of SP-B protein, decreased levels of SP-B may increase susceptibility to lung injury and/or contribute directly to altered lung structure and function. Disruption of one SP-B allele in mice (SP-B+/- mice) resulted in 50% reduction in SP-B mature peptide in bronchoalveolar lavage fluid (BALF) (7): SP-B(+/-) mice exhibited decreased lung compliance, air trapping at low lung volumes, and significantly increased lung injury in response to hyperoxia (5, 7, 8). Mutations in the SP-B gene associated with partial SP-B deficiency (9, 10) or transient SP-B deficiency (11) were linked to development of chronic lung disease in human infants. Decreased levels of SP-B in BALF were detected in patients at risk to develop acute respiratory distress syndrome (12). SP-B deficiency was also detected following infection with adenovirus (13), respiratory syncytial virus (14), Pneumocytis carinii (15, 16), or treatment of mice with endotoxin (17, 18). Whether the decrease in SP-B protein concentration contributed to the severity of lung inflammation and injury following infection remains an important and unanswered question.

Although SP-B deficiency may predispose to lung injury, it is unclear whether increased levels of SP-B have a protective effect on lung function during injury. Pretreatment of SP-B(+/-) mice with SP-B corrected the oxygen-induced surfactant dysfunction (19). Exposure of wild-type mice to aerosolized endotoxin resulted in lung inflammation and decreased levels of SP-B coincident with surfactant dysfunction (17). Addition of a mixture of SP-B and SP-C to surfactant isolated from endotoxin-exposed mice significantly improved the surface properties of isolated surfactant in vitro (17). The present study was undertaken to assess whether genetically increased levels of SP-B would improve lung function and diminish lung inflammation following intratracheal endotoxin exposure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Three groups of mice were used for the current studies: SP-B heterozygous mice (SP-B+/-), wild-type mice (SP-B+/+), and transgenic mice expressing human SP-B (SP-B–overexpressing mice or SP-B+/+/+). Generation of SP-B(+/-) and SP-B(+/+/+) mice has previously been described (6, 20, 21). To eliminate differences due to genetic background, all mice used in the current study were offspring of sibling matings of SP-B(+/-) mice carrying the human SP-B transgene. The concentration of mature SP-B peptide in BALF was decreased by 50% in SP-B(+/-) mice and increased by ~ 250% in SP-B(+/+/+) mice, compared with wild-type littermates (20). The three SP-B genotypes were indistinguishable with respect to birth weight, lung weight, lung structure, somatic growth, and reproductive capacity. All mice were housed under pathogen-free conditions and were studied under a protocol approved by the Institutional Animal Care and Use Committee. For each experiment, 5- to 6-wk-old (20–25 g body weight) littermates were used.

Endotoxin Treatment
Mice were anesthetized with isofluorane, and 0.5 µg of Escherichia coli F583 (Sigma Chemical Co, St. Louis, MO), suspended in 100 µl endotoxin-free phosphate-buffered saline (PBS), was administered intratracheally just below the corticoid cartilage. Untreated control mice and mice treated identically with an equal volume of vehicle were also evaluated. At various time points after treatment, mice were anesthetized with intraperitoneal pentobarbital, and the lungs lavaged with three 1-ml aliquots of PBS. Aliquots of BALF containing protease inhibitor cocktail (Sigma) were stored at -70°C until analysis. For cell counts, an aliquot of BALF was centrifuged at 250 x g for 10 min, and the cell pellet washed in red blood cell lysis buffer (0.1 M, NH4Cl in 0.1 M Tris pH 7.6, 0.1 mM EDTA) and resuspended in PBS for quantitative analysis by hemocytometry. Differential cell counts were made on cytospin preparations stained with May-Grünwald Giemsa stain (Diff-Quik; Scientific Products, McGraw Park, IL). Protein concentration in BALF was determined by the bicinchoninic assay (22). Aliquots of BALF containing 0.5 µg protein were analyzed by Western blotting using antibodies directed against SP-A (23), SP-D (24), or SP-C (25). The level of mature SP-B peptide in BALF was quantitated by enzyme-linked immunosorbent assay (ELISA) (20). Cytokine levels in BALF were measured by sandwich immunoassay (R&D Systems, Minneapolis, MN). All samples were assayed in duplicate. Differences between groups were assessed by one-way ANOVA, and differences between means were assessed by contrast comparison and Student-Newman-Keuls test (StatView; SAS Institute Inc., Cary, NC).

Lung Mechanics
Twenty-four hours after endotoxin treatment, mice (n = 4/group) were anesthetized (intraperitoneally) with 0.1 ml/10 g body weight of a mixture containing 40 mg/ml ketamine and 2 mg/ml xylazine. Mice were tracheostomized and ventilated with a tidal volume of 8 ml/kg at a rate of 450 breaths/min and positive end-expiratory pressure of 2 cm H2O by a computerized Flexi Vent system (SCIREQ, Montreal, PQ, Canada) (26). This machine features accurate measurement of volume by using the position of the ventilator piston and pressure in the cylinder, and allows analysis of dynamic lung compliance in mice. The ventilation mode on the Flexi Vent was changed to forced oscillatory signal (0.5–19.6 Hz), and respiratory impedance was measured. Estimated tissue damping and tissue elastance for mice at 2 cm H2O PEEP were obtained by fitting a model to each impedance spectrum. With this system, the calibration procedure removed the impedance of the equipment and tracheal tube.

Surfactant Function
Large aggregate surfactant was isolated by centrifugation, and surfactant activity measured with a captive bubble surfactometer (27, 28). The concentration of each sample was adjusted to 3.6 nmol/µl saturated phosphatidylcholine, and 3 µl of surfactant was applied to the air–water interface of a 26-µl volume bubble by microsyringe (n = 3/group). Sensitivity to protein inhibition was assessed in the presence of 5 or 10% sheep plasma protein (vol/vol). Surface tension was measured every 10 s for 300 s to establish equilibrium surface tension, then bubble pulsation was started. The minimum surface tension after 65% bubble volume reduction was measured at the fifth pulsation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Endotoxin on Alveolar SP-B Levels
SP-B peptide levels in BALF from SP-B(+/+/+) mice were increased by 5.6-fold relative to SP-B(+/-) mice, and 3-fold compared with SP-B(+/+) FVB/N mice, consistent with our previous finding (Figure 1) (20). Mature SP-B peptide levels in BALF were quantitated by ELISA at 3, 6, 24, 48, and 72 h after intratracheal exposure to endotoxin. Endotoxin inhibited SP-B concentrations, which reached minimal levels at 6 or 24 h after treatment (Figure 1). In SP-B(+/-) mice, SP-B concentrations were decreased 51% at 24 h after treatment. SP-B levels in SP-B(+/+) and SP-B(+/+/+) mice were decreased 36 and 18%, respectively, 24 h after treatment. Total protein levels in BALF were increased in all groups of mice, but were significantly increased in SP-B (+/-) mice compared with SP-B(+/+) and SP-B(+/+/+) mice at 24 h after treatment (Figure 2). Complete recovery of SP-B levels and restoration of protein levels in BALF was observed in all groups 72 h after treatment. These results indicate that the effect of endotoxin on protein leak and SP-B concentration in BALF was more severe in SP-B(+/-) mice compared with SP-B(+/+/+) and SP-B(+/+) mice.



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  		Figure 1. Levels of SP-B in BALF. Five-week-old SP-B(+/+), SP-B(+/-), or SP-B(+/+/+) mice were injected intratracheally with 0.5 µg of endotoxin (n = 5 for each time point). At 3, 6, 24, 48, and 72 h after injection, BALF was obtained from 4–5 mice, and SP-B levels measured by ELISA. Control groups included uninjected mice and mice injected with PBS. SP-B levels in injected controls were not significantly different from uninjected controls; only the 24-h time point is shown for the PBS-injected control group. *P < 0.001, **P < 0.01, relative to SP-B(+/+/+) mice. *tP < 0.02, relative to PBS-injected mice of same genotype.

 


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  		Figure 2. Total protein in BALF. Levels of total protein in BALF were assessed in five-week-old SP-B(+/+), SP-B(+/-), and SP-B(+/+/+) mice 3, 6, 24, 48, and 72 h after intratracheal injection of 0.5 µg endotoxin (n = 5 for each time point). Control groups included uninjected mice and mice injected with PBS. SP-B levels in injected controls were not significantly different from uninjected controls; only the 24-h time point is shown for the PBS-injected control group. *P < 0.02, relative to PBS-injected mice of same genotype; *tP < 0.04 relative to SP-B(+/+) and SP-B(+/+/+) mice at 24 h.

 
Pulmonary Inflammation following Endotoxin Exposure
The effect of genetically altered SP-B levels on endotoxin-induced lung inflammation was assessed by measuring recruitment of cells into the airspaces at 6 h after treatment (Figure 3). Total cell counts in BALF were significantly lower in SP-B(+/+/+) mice and SP-B(+/+) mice compared with SP-B(+/-) mice. The influx of neutrophils was significantly lower in SP-B(+/+/+) mice compared with SP-B(+/+) mice or SP-B(+/-) mice. As an additional index of pulmonary inflammation, levels of selected cytokines in BALF were assessed 6 h after intratracheal injection of endotoxin (Figure 4). Interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, and macrophage inflammatory protein-2 were significantly lower in SP-B(+/+/+) mice compared with SP-B(+/-) mice following endotoxin challenge, whereas KC was significantly lower in both SP-B(+/+/+) and SP-B(+/+) mice compared with SP-B(+/-) mice. IL-1 and IL-10 were similarly increased in all groups following endotoxin exposure (not shown). Cytokines were not detectable in BALF from uninjected mice or mice that were intratracheally injected with PBS (not shown). Levels of SP-A, SP-D, and SP-C in BALF were similar in all groups of mice 6 h after intratracheal injection of endotoxin (Figure 5). Nitrite levels in BALF were also similar among the three groups of mice at 6 h after treatment, and were not significantly different from uninfected mice (not shown). Taken together, these results indicate that SP-B decreased inflammation following intratracheal administration of endotoxin.



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  		Figure 3. Cell counts in BALF. Five-week-old SP-B(+/+), SP-B(+/-), and SP-B(+/+/+) mice were injected intratracheally with 0.5 µg of endotoxin (n = 5 for each group). Twenty-four hours after infection, BALF was collected for analysis of total and differential cell counts. *P < 0.02, **P < 0.03 relative to SP-B(+/-) mice.

 


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  		Figure 4. Inflammatory mediators in BALF. Levels of mTNF-{alpha}, KC, mIL-6, and MIP-2 in BALF were assessed 6 h after intratracheal instillation of 0.5 µg of endotoxin to SP-B(+/+), SP-B(+/-), and SP-B(+/+/+) mice (n = 5 for each group). Levels of inflammatory mediators in BALF from uninjected mice were below the detection limit. *P < 0.05 relative to SP-B(+/+) and SP-B(+/+/+) mice.

 


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  		Figure 5. SP-A, SP-D and SP-C in BALF. Five-week-old SP-B(+/+), SP-B(+/-), and SP-B(+/+/+) mice were injected intratracheally with 0.5 µg of endotoxin (n = 4 for each group). BALF containing 0.5 µg protein was subjected to SDS-PAGE/Western blotting, and the levels of SP-A, SP-D, and SP-C assessed by scanning densitometry. Results are expressed as percentage of uninfected SP-B(+/+) mice.

 
Lung Mechanics
Lung compliance was significantly decreased by endotoxin instillation in SP-B(+/+) and SP-B(+/-) mice (Figure 6). In contrast, lung compliance was not altered by endotoxin in SP-B(+/+/+) mice. Tissue damping and tissue elastance were increased 7- to 8-fold by endotoxin in SP-B(+/+) and SP-B(+/-) mice (Figure 6). In SP-B(+/+/+) mice, tissue damping was not altered by endotoxin instillation, and the change in tissue elastance was less than that in the other genotypes, with only a 1.4-fold increase. Lung mechanics were altered by endotoxin in both SP-B(+/+) and SP-B(+/-) mice, whereas SP-B(+/+/+) mice showed minimal changes in lung mechanics or no changes at all.



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  		Figure 6. Lung mechanics. Five-week-old SP-B(+/+), SP-B(+/-), and SP-B(+/+/+) mice were injected intratracheally with 0.5 µg of endotoxin (n = 4 for each group). Compliance, tissue damping, and tissue elastance were measured on anesthetized mice 24 h after endotoxin treatment. *P < 0.05 relative to PBS-treated mice of the same genotype.

 
Surfactant Function
The surface activities of large aggregate surfactant isolated from BALF of unchallenged SP-B(+/-), SP-B(+/+), and SP-B(+/+/+) mice were compared using a captive bubble surfactometer. Equilibrium surface tensions of surfactant obtained from the three groups of mice were comparable (not shown). Minimum surface tension of surfactant was low for all three groups, but was significantly lower in SP-B(+/+/+) mice compared with SP-B(+/-) and SP-B(+/+) mice. In the presence of 5% plasma protein, low surface tension was maintained by surfactant from SP-B(+/+/+) and SP-B(+/+) but not SP-B(+/-) mice. Minimum surface tension in the presence of 10% plasma protein was increased for all three groups, but the increase was significantly lower for surfactant from SP-B(+/+/+) mice, suggesting that surfactant from these mice is more resistant to plasma protein mediated surfactant dysfunction (Figure 7).



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  		Figure 7. Surfactant function. BALF was obtained from 5-wk-old unchallenged SP-B(+/+), SP-B(+/-), and SP-B(+/+/+) mice (n = 5 for each group). The large aggregate fraction of surfactant was isolated, pooled for each group, and equilibrium surface tension (not shown) and minimum surface activity assessed by captive bubble surfactometry. Minimum surface tension was also assessed following the addition of 5 or 10% (vol/vol) plasma protein. *P < 0.05 relative to PBS-treated mice of the same genotype.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SP-B expression was genetically manipulated to generate transgenic mouse lines in which SP-B concentrations in BALF varied without changing alveolar surfactant pool sizes or surfactant phospholipid composition (7, 21). In a previous study, elevated concentration of SP-B in BALF did not increase bacterial killing in SP-B(+/+/+) mice, and decreased levels of SP-B in SP-B(+/-) mice did not increase susceptibility to infection (20); however, the effect of SP-B levels on inflammation association with infection was not assessed in these mice. In the present study, endotoxin-induced lung inflammation, assessed by influx of neutrophils and elaboration of proinflammatory cytokines in the airspaces, was abrogated in mice expressing higher levels of SP-B peptide. Increased concentration of SP-B in BALF was also associated with significantly improved lung function in endotoxin-challenged mice. Taken together, these results suggest that SP-B is not an antibacterial peptide, but may play an important role in protecting against lung inflammation arising from bacterial infection.

It is not clear if the protective effect of SP-B is related to an anti-inflammatory property of the peptide. Miles and coworkers (29) reported that SP-B inhibited endotoxin-induced nitric oxide production by isolated alveolar macrophages, consistent with a direct anti-inflammatory effect. However, in the present study, nitrite levels were similar in SP-B(+/-) and SP-B(+/+/+) mice, suggesting that nitric oxide levels were not influenced by SP-B following treatment with endotoxin. Elevated levels of mature SP-B peptide were, however, associated with enhanced surfactant function (lower minimum surface tension) and increased resistance of surfactant to inhibition by plasma proteins in surfactant from unchallenged mice and lower levels of protein in BALF following endotoxin challenge in vivo. The effect of endotoxin on lung function, as assessed by compliance, tissue damping, and tissue elastance, was significantly greater in SP-B(+/+) and SP-B(+/-) mice compared with SP-B(+/+/+) mice, consistent with a protective effect of SP-B. Although these data do not exclude a direct anti-inflammatory effect of SP-B, they do suggest that SP-B may indirectly protect against endotoxin-induced lung inflammation by reducing the severity of lung injury in SP-B(+/+/+) mice, resulting in decreased influx of inflammatory cells.

An alternative explanation for decreased inflammation and improved lung function in endotoxin-treated SP-B(+/+/+) mice is that SP-B directly or indirectly inhibits endotoxin action. Alveolar surfactant pool size and surfactant phospholipid composition were not significantly different among the SP-B(+/-), SP-B(+/+), and SP-B(+/+/+) groups, suggesting that quantitative or qualitative differences in surfactant phospholipids could not account for the diminished effect of endotoxin in SP-B–overexpressing mice. Surfactant proteins, including SP-A (30), SP-D (31), and SP-C (32, 33), bind to endotoxin; however, the concentration of each of those surfactant proteins was not altered by over- or underexpression of SP-B (Figure 5). It is unlikely that SP-B binds to endotoxin, because it was recently reported that SP-C was the only hydrophobic component of surfactant that bound endotoxin. Given these results, it seems likely that the protective effect of SP-B is mediated through enhanced surfactant function rather than inhibition of endotoxin action; however, we cannot exclude the possibility that the concentration of an unknown endotoxin-binding component in BALF was altered by elevated expression of SP-B.

Although elevated levels of SP-B in overexpressing mice were associated with a protective effect, SP-B(+/-) mice were significantly more susceptible to endotoxin-induced lung inflammation than either SP-B(+/+) or SP-B(+/+/+) mice. This observation is consistent with previous reports that lung function is more severely compromised in SP-B(+/-) mice than in SP-B(+/+) littermates during exposure to hyperoxia (8, 19). Intratracheal administration of endotoxin resulted in markedly lower levels of mature SP-B peptide in BALF from each group; however, the magnitude of the decrease was greatest in SP-B(+/-) mice and was least in the SP-B(+/+/+) group. An inhibitory effect of endotoxin on SP-B expression has previously been reported. Exposure of 129J mice to aerosolized endotoxin resulted in decreased SP-B mRNA and mature peptide without significant change in SP-A levels (17). Intratracheal administration of endotoxin to FVB/N mice resulted in decreased staining of alveolar epithelial cells for SP-B and SP-C proproteins, although staining for SP-B mature peptide was increased in the airspaces (18). In the present study, intratracheal administration of endotoxin to FVB/N mice resulted in decreased levels of mature SP-B peptide in BALF at 6 h after treatment without significant changes in SP-A, SP-D, or SP-C concentrations, consistent with a specific effect of endotoxin on SP-B expression. The effect of endotoxin on SP-B expression and SP-B levels in BALF was likely mediated by the type II cell, because the SP-B proprotein is processed to the mature peptide by type II cells but not Clara cells (34).

The effects of endotoxin on pulmonary inflammation are mediated in part through TNF-{alpha} (35, 36). Intratracheal administration of TNF-{alpha} to FVB/N mice resulted in decreased levels of SP-B mRNA, suggesting that this pathway may contribute to surfactant dysfunction following infection (37). In the present study, TNF-{alpha} levels were highest in BALF of SP-B(+/-) mice. The fact that this group also had the greatest decrease in mature SP-B peptide levels following endotoxin exposure suggests that this cytokine may partly account for the increased susceptibility of SP-B(+/-) mice to endotoxin-induced lung inflammation. It remains unclear if other cytokines secreted in response to endotoxin treatment contribute to this effect.

In summary, elevated levels of mature SP-B peptide in the airspaces of transgenic mice were associated with decreased inflammation following exposure to endotoxin. The protective effect of SP-B was likely mediated through enhanced surfactant function, leading to reduced lung injury, decreased influx of inflammatory cells, and lower cytokine levels. Endotoxin treatment of SP-B(+/-) mice resulted in decreased SP-B peptide levels, which likely exacerbated lung injury in this group. Collectively, these results suggest that infection superimposed on SP-B deficiency leads to a significantly worse outcome; in contrast, elevated levels of SP-B peptide, in the absence of increased surfactant phospholipids or other surfactant proteins, inhibits endotoxin-induced lung inflammation.


   Acknowledgments
 
The secretarial assistance of Ms. Ann Maher and technical assistance of William M. Hull are gratefully acknowledged. This study was supported by grants from the National Heart, Lung and Blood Institute, R37-HL56285 (T.E.W.), P01-HL61646 (M.I., J.A.W., A.H.J., T.E.W.) and R37-HL38859 (J.A.W.).

Received in original form May 17, 2002

Received in final form September 11, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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