Ann Marie LeVine

Distinct Effects of Surfactant Protein A or D Deficiency During Bacterial Infection on the Lung

Mice lacking surfactant protein (SP)-A (SP-A−/−) or SP-D (SP-D−/−) and wild-type mice were infected with group B streptococcus or Haemophilus influenzae by intratracheal instillation. Although decreased killing of group B streptococcus and H. influenzae was observed in SP-A−/− mice but not in SP-D−/− mice, deficiency of either SP-A or SP-D was associated with increased inflammation and inflammatory cell recruitment in the lung after infection. Deficient uptake of bacteria by alveolar macrophages was observed in both SP-A- and SP-D-deficient mice. Isolated alveolar macrophages from SP-A−/− mice generated significantly less, whereas those from SP-D−/− mice generated significantly greater superoxide and hydrogen peroxide compared with wild-type alveolar macrophages. In SP-D−/− mice, bacterial killing was associated with increased lung inflammation, increased oxidant production, and decreased macrophage phagocytosis. In contrast, in the absence of SP-A, bacterial killing was decreased and associated with increased lung inflammation, decreased oxidant production, and decreased macrophage phagocytosis. Increased oxidant production likely contributes to effective bacterial killing in the lungs of SP-D−/− mice. The collectins, SP-A and SP-D, play distinct roles during bacterial infection of the lung.

Surfactant protein-A enhances respiratory syncytial virus clearance in vivo

To determine the role of surfactant protein-A(SP-A) in antiviral host defense, mice lacking SP-A (SP-A-/-) were produced by targeted gene inactivation. SP-A-/- and control mice (SP-A+/+) were infected with respiratory syncytial virus (RSV) by intratracheal instillation. Pulmonary infiltration after infection was more severe in SP-A-/- than in SP-A+/+ mice and was associated with increased RSV plaque-forming units in lung homogenates. Pulmonary infiltration with polymorphonuclear leukocytes was greater in the SP-A-/- mice. Levels of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 were enhanced in lungs of SP-A-/- mice. After RSV infection, superoxide and hydrogen peroxide generation was deficient in macrophages from SP-A-/- mice, demonstrating a critical role of SP-A in oxidant production associated with RSV infection. Coadministration of RSV with exogenous SP-A reduced viral titers and inflammatory cells in the lung of SP-A-/- mice. These findings demonstrate that SP-A plays an important host defense role against RSV in vivo.

Surfactant Protein D Enhances Clearance of Influenza A Virus from the Lung In Vivo

Mice lacking surfactant protein surfactant protein D (SP-D−/−) and wild-type mice (SP-D+/+) were infected with influenza A virus (IAV) by intranasal instillation. IAV infection increased the endogenous SP-D concentration in wild-type mice. SP-D-deficient mice showed decreased viral clearance of the Phil/82 strain of IAV and increased production of inflammatory cytokines in response to viral challenge. However, the less glycosylated strain of IAV, Mem/71, which is relatively resistant to SP-D in vitro, was cleared efficiently from the lungs of SP-D−/− mice. Viral clearance of the Phil/82 strain of IAV and the cytokine response were both normalized by the coadministration of recombinant SP-D. Since the airway is the usual portal of entry for influenza A virus and other respiratory pathogens, SP-D is likely to play an important role in innate defense responses to IAV.

Pulmonary collectins and innate host defense of the lung.

Surfactant proteins A and D (SP-A and SP-D) are members of the collectin family of polypeptides expressed in the respiratory tract that bind bacterial, fungal and viral pathogens, enhancing their opsonization and killing by phagocytic cells. Clearance of bacterial pathogens including group B streptococci, Haemophilus influenza, Pseudomonas aeruginosa and viral pathogens, respiratory syncytial virus, adenovirus and influenza A virus, was deficient in SP-A(-/-) mice. SP-A deficiency was associated with enhanced inflammation and synthesis of proinflammatory cytokines. SP-D(-/-) mice cleared these bacteria as efficiently as wild-type mice; however, clearance of viral pathogens was deficient in SP-D(-/-) mice and associated with increased inflammation. SP-A and SP-D play critical and distinct roles in the regulation of alveolar macrophage function and inflammation, contributing to innate defense of the lung.

GM-CSF–deficient mice are susceptible to pulmonary group B streptococcal infection

Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-targeted mice (GM-/-) cleared group B streptococcus (GBS) from the lungs more slowly than wild-type mice. Expression of GM-CSF in the respiratory epithelium of GM-/- mice improved bacterial clearance to levels greater than that in wild-type GM+/+ mice. Acute aerosolization of GM-CSF to GM+/+ mice significantly enhanced clearance of GBS at 24 hours. GBS infection was associated with increased neutrophilic infiltration in lungs of GM-/- mice, while macrophage infiltrates predominated in wild-type mice, suggesting an abnormality in macrophage clearance of bacteria in the absence of GM-CSF. While phagocytosis of GBS was unaltered, production of superoxide radicals and hydrogen peroxide was markedly deficient in macrophages from GM-/- mice. Lipid peroxidation, assessed by measuring the isoprostane 8-iso-PGF2alpha, was decreased in the lungs of GM-/- mice. GM-CSF plays an important role in GBS clearance in vivo, mediated in part by its role in enhancing superoxide and hydrogen peroxide production and bacterial killing by alveolar macrophages.

Surfactant content in children with inflammatory lung disease.

OBJECTIVE To determine surfactant profiles of tracheal secretions in mechanically ventilated children with respiratory failure secondary to bacterial pneumonia, viral pneumonitis, adult respiratory distress syndrome (ARDS), and cardiopulmonary bypass. DESIGN Prospective, cohort study. SETTING Tertiary, multidisciplinary, pediatric intensive care unit. PATIENTS One hundred twenty pediatric patients with respiratory failure requiring mechanical ventilation. INTERVENTIONS Routine tracheal aspirates were collected from children with bacterial pneumonia, viral pneumonitis, ARDS, postcardiopulmonary bypass, and a postsurgical control group. Samples were obtained on days 1, 2, 3, after every week of intubation and on the day of extubation. MEASUREMENTS AND MAIN RESULTS The tracheal aspirates were analyzed by high-performance liquid chromatography for lecithin/sphingomyelin rations and by enzyme-linked immunosorbent assay for surfactant proteins A and B. Lung compliance and the oxygenation index were measured on each day of sample collection. On day 1, patients with bacterial pneumonia, viral pneumonitis, and ARDS had decreased lecithin/sphingomyelin ration (p < .001), and those patients with bacterial pneumonia and viral pneumonitis had decreased surfactant protein A/protein concentration (p < .001). The lecithin/sphingomyelin ratios and surfactant protein A/protein concentration were significantly different among the groups (p < .001), with the bacterial pneumonia and viral pneumonitis groups having higher lecithin/sphingomyelin ratios and increased surfactant protein concentrations before extubation. Pulmonary compliance was lower and the oxygenation index was higher than controls (p < .001) in patients with bacterial pneumonia, viral pneumonitis, and ARDS. Pulmonary compliance was correlated weakly with lecithin/sphingomyelin ratio (r2 = .11, p < .001) and surfactant protein A/protein concentration (r2 = .03, p < .05). Surfactant protein B was similar in the diagnostic groups. Surfactant content in tracheal secretions from cardiopulmonary bypass patients was equivalent to controls. CONCLUSION Abnormal tracheal aspirate surfactant phospholipids and surfactant protein A were noted in children with bacterial pneumonia, viral pneumonitis, and ARDS, but not in children on cardiopulmonary bypass.

Surfactant Protein-D Regulates Soluble CD14 through Matrix Metalloproteinase-12

Surfactant protein D (SP-D) and CD14 are important innate immune defense molecules that mediate clearance of pathogens and apoptotic cells from the lung. To test whether CD14 expression and function were influenced by SP-D, the surface expression of CD14 was assessed on alveolar macrophages from SP-D−/− mice. CD14 was reduced on alveolar macrophages from SP-D−/− mice and was associated with reduced uptake of LPS and decreased production of TNF-α after LPS stimulation. CD14 is proteolytically cleaved from the cell surface to form a soluble peptide. Soluble CD14 (sCD14) was increased in the bronchoalveolar lavage fluid from SP-D−/− mice. Because matrix metalloproteinase (MMP)-9 and -12 activities were increased in the lungs of SP-D−/− mice, the role of these metalloproteases in the production of sCD14 was assessed. sCD14 was decreased in both MMP9−/−/SP-D−/− and MMP12−/−/SP-D−/− mice demonstrating MMP-9 and MMP-12 contribute to proteolytic shedding of CD14. The increased sCD14 seen in SP-D−/− mice was dependent upon the activation of MMP-12 via an MMP-9-dependent mechanism. Supporting this observation, MMP-12 caused the release of sCD14 from RAW 264.7 cells in vitro. In conclusion, SP-D influences innate host defense, in part, by regulating sCD14 in a process mediated by MMP-9 and MMP-12.

Effect of Lung Surfactant Collectins on Bronchoalveolar Macrophage Interaction with Blastomyces dermatitidis: Inhibition of Tumor Necrosis Factor Alpha Production by Surfactant Protein D

ABSTRACT Alveolar surfactant modulates the antimicrobial function of bronchoalveolar macrophages (BAM). Little is known about the effect of surfactant-associated proteins in bronchoalveolar lavage fluid (BALF) on the interaction of BAM and Blastomyces dermatitidis. We investigated BALF enhancement or inhibition of TNF-α production by BAM stimulated by B. dermatitidis. BAM from CD-1 mice were stimulated with B. dermatitidis without or with normal BALF, surfactant protein A-deficient (SP-A−/−) or surfactant protein D-deficient (SP-D−/−) BALF, or a mixture of SP-A−/− and SP-D−/− BALF. An enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha (TNF-α) in culture supernatants. BALFs were standardized in protein concentration. BAM plus B. dermatitidis (BAM-B. dermatitidis) TNF-α production was inhibited ≥47% by BALF or SP-A−/− BALF (at 290 or 580 μg of protein/ml, P < 0.05 to 0.01); in contrast, SP-D−/− BALF did not significantly inhibit TNF-α production. If SP-A−/− BALF was mixed in equal amounts with SP-D−/− BALF, TNF-α production by BAM-B. dermatitidis was inhibited (P < 0.01). Finally, pure SP-D added to SP-D−/− BALF inhibited TNF-α production by BAM-B. dermatitidis (P < 0.01). B. dermatitidis incubated with BALF and washed, plus BAM, stimulated 63% less production of TNF-α than did unwashed B. dermatitidis (P < 0.05). SP-D was detected by anti-SP-D antibody on BALF-treated unwashed B. dermatitidis in an immunofluorescence assay (IFA). The BALF depleted by a coating of B. dermatitidis lost the ability to inhibit TNF-α production (P < 0.05). 1,3-β-Glucan was a good stimulator of BAM for TNF-α production and was detected on B. dermatitidis by IFA. β-Glucan incubated with BALF inhibited the binding of SP-D in BALF to B. dermatitidis as demonstrated by IFA. Our data suggest that SP-D in BALF binds β-glucan on B. dermatitidis, blocking BAM access to β-glucan, thereby inhibiting TNF-α production. Thus, whereas BALF constituents commonly mediate antimicrobial activity, B. dermatitidis may utilize BALF constituents, such as SP-D, to blunt the host defensive reaction; this effect could reduce inflammation and tissue destruction but could also promote disease.

Surfactant biology and clinical application

There is strong evidence that alterations in the pulmonary surfactant system play an important role in the pathophysiology of lung disease, including ARDS . Although it is still unclear whether mortality and morbidity of ARDS will be reduced, surfactant replacement therapy has been shown to improve oxygenation, improve lung compliance, and decrease the need for ventilatory support. The critical need for more standardized studies with one type of intratracheal surfactant and uniform measurements of surfactant proteins and phospholipids by BAL is evident. Further studies will also be needed to elucidate the optimal timing and dosage regimen for different disease processes. Some evidence supports the measurements of surfactant protein levels as markers for predicting the onset and outcome of ARDS and perhaps providing a window for early treatment of patients at risk to develop ARDS. Continued investigation into the role of surfactant in the immune regulation of the lung may also provide additional information to support the efficacy of surfactant replacement in lung disease.