Surfactant protein D reduces alveolar macrophage apoptosis in vivo.

Surfactant protein D (SP-D) is a molecule of the innate immune system that recognizes the patterns of surface carbohydrate on pathogens and targets them for phagocytosis and killing. SP-D-deficient mice show an increased number of macrophages in the alveolar space, excess surfactant phospholipid, overproduction of reactive oxygen species, and the development of emphysema. We report here that SP-D-deficient mice have a 5- to 10-fold increase in the number of apoptotic and necrotic alveolar macrophages, as defined by annexin V and propidium iodine staining, respectively. Intrapulmonary administration of a truncated 60-kDa fragment of human recombinant SP-D reduces the number of apoptotic and necrotic alveolar macrophages and partially corrects the lipid accumulation in SP-D-deficient mice. The same SP-D fragment binds preferentially to apoptotic and necrotic alveolar macrophages in vitro, suggesting that SP-D contributes to immune homeostasis in the lung by recognizing and promoting removal of necrotic and apoptotic cells.

Lymphocyte activation in the lungs of SP-D null mice.

Surfactant protein D (SP-D) appears to play an important role in regulating local pulmonary inflammatory responses to pathogens. There is also in vitro evidence that SP-D may suppress local T cell responses. However, the role of SP-D in regulating T cell responses directly in the lung has not been previously evaluated in vivo. SP-D(-)(/-) mice demonstrate peribronchial and perivascular accumulations of lymphocytes. Therefore, we investigated the functional status and abundance of intrapulmonary lymphocytes in SP-D(-)(/-) mice. By morphometric analysis, SP-D(-)(/-) mice demonstrated increased numbers of airway- and vessel-associated lymphocytes without increases in interstitial lymphocytes. There was increased proliferative activity of lymphocytes isolated by enzymatic disassociation of minced lung. Flow cytometry was used to determine the number and functional activation status of intrapulmonary CD4(+) and CD8(+) T cells, as well as B cells and NK cells. Cytokine expression patterns in lung tissues were evaluated using RNase protection assays, reverse transcriptase/polymerase chain reaction, and enzyme-linked immunosorbent assay. There was marked T cell activation in the lungs of SP-D(-)(/-) mice, as reflected by an increased percentage of both CD4(+) and CD8(+) T cells expressing CD69 and CD25. BAL CD4 lymphocytes were increased and the fraction expressing CD69 was also increased. Although there were increases in BAL CD8 lymphocytes, apparent increases in CD69-positive CD8 lymphocytes did not reach statistical significance. In contrast, splenic T cells were not activated in SPD(-)(/-) mice. Of the proinflammatory cytokines evaluated, only interleukin (IL)-12 and IL-6 expression were consistently upregulated in the lungs of SPD(-)(/-) mice. Increased IL-2 expression was apparent but did not reach statistical significance. We conclude that the lack of local pulmonary production of SP-D leads to a state of persistent T cell activation, possibly in response to exogenous antigens. This study therefore provides further evidence of the important local immunoregulatory role of SP-D in vivo.

Continuous positive airway pressure and surfactant; combined data from animal experiments and clinical trials.

Bronchopulmonary dysplasia (BPD) remains a cause of considerable morbidity for the preterm infant. Ventilation is a primary risk factor. This review discusses the rationale for combining surfactant and nasal continuous positive airway pressure (nCPAP) using evidence from both clinical and animal studies. The early application of nCPAP with or without surfactant is safe and reduces the need for mechanical ventilation. Combining nCPAP with surfactant results in dramatically improved lung structure in a primate model of BPD, but still does not allow for normal alveolarization. BPD is a complex condition resulting from the interaction of many factors. Experimental evaluation of nCPAP in appropriate animal models will allow new strategies for prevention and treatment of BPD to be developed.

SP-A-enriched surfactant for treatment of rat lung transplants with SP-A deficiency after storage and reperfusion.

BACKGROUND: The function of pulmonary surfactant is affected by lung transplantation, contributing to impaired lung transplant function. A decreased amount of surfactant protein-A (SP-A) after reperfusion is believed to contribute to the impaired surfactant function. Surfactant treatment has been shown to improve lung transplant function, but the effect is variable. We investigated whether SP-A enrichment of surfactant improved the efficacy of surfactant treatment in lung transplantation. METHODS: Left and right lungs of Lewis rats, inflated with 50% O2, were stored for 20 hr at 8 degrees C. Surfactant in bronchoalveolar lavage fluid from right lungs was investigated after storage (n=6). Left lungs were transplanted into syngeneic recipients and treated with SP-A-deficient surfactant (n=6) or SP-A-enriched surfactant (n=6) just before reperfusion. Air was instilled into untreated lung transplants (n=6). Sham operated (n=4) and normal (n=8) animals served as controls. Lung function was measured during 1 hr of reperfusion; surfactant components in bronchoalveolar lavage fluid were measured after reperfusion. RESULTS: After storage the amount of SP-A decreased by 27%, whereas surfactant phospholipids changed minimally. After reperfusion a further decrease of SP-A was paralleled by profound changes in surfactant phospholipids. Lung transplant function, however, remained relatively good. After instillation of SP-A-enriched surfactant, PO2 values were reached that approximated sham control PO2 values, whereas after SP-A-deficient surfactant treatment, the PO2 values did not improve. CONCLUSION: Enrichment of surfactant with SP-A for treatment of lung transplants improves the efficacy of surfactant treatment.

Surfactant protein-A--deficient mice display an exaggerated early inflammatory response to a beta-resistant strain of influenza A virus.

Surfactant protein (SP)-A is a member of the collectin family of proteins. In vitro, SP-A binds influenza A virus (IAV), neutralizes infectivity, and enhances uptake by macrophages. SP-D also binds and neutralizes certain strains of IAV. To determine if SP-A has a role in protecting the intact animal against IAV infection, we inoculated gene-targeted SP-A-deficient mice (-/-) and littermate controls (+/+) with either saline or increasing doses of an IAV strain that binds SP-A but not SP-D. IAV was more virulent in SP-A-/- compared with +/+ mice, with a significantly lower mean lethal dose (LD(50)) and significantly greater weight loss during infection. SP-A-/- mice also had increased airway epithelial injury and more alveolar cellular infiltrates than +/+ mice. On Day 2, SP-A-/- mice had more neutrophils and higher MIP-2 levels in the lung than +/+ mice. We conclude the altered host response and increased susceptibility to X-79Delta167 infection in SP-A-/- mice reflects a protective role for SP-A in regulating the host response to IAV. Because the recovery of virus from lung homogenates on Days 2 and 6 after inoculation was comparable in -/- and +/+ mice, we speculate SP-A reduces IAV virulence independently of direct viral neutralization.

Deficiency of SP-B reveals protective role of SP-C during oxygen lung injury.

Although the surface properties of surfactant protein (SP)-B and SP-C are similar, the contributions that either protein may make to lung function have not been identified in vivo. Mutations in SP-B cause lethal respiratory failure at birth; however, SP-B null mice are deficient in both SP-B and SP-C. To identify potential contributions of SP-C to lung function in vivo, the following transgenic mice were generated and exposed to 95% O(2) for 3 days: (SP-B(+/+),SP-C(+/+)), (SP-B(+/+), SP-C(-/-)), (SP-B(+/-),SP-C(+/+)), (SP-B(+/-),SP-C(+/-)), and (SP-B(+/-),SP-C(-/-)). Hyperoxia altered pressure-volume curves in mice that were heterozygous for SP-B, and these values were further decreased in (SP-B(+/-),SP-C(-/-)) mice. Likewise, alveolar interleukin (IL)-6 and IL-1 beta were maximally increased by O(2) exposure of (SP-B(+/-),SP-C(-/-)) mice compared with the other genotypes. Lung hysteresivity was lower in the (SP-B(+/-),SP-C(-/-)) mice. Surfactant isolated from (SP-B(+/+),SP-C(-/-)) and (SP-B(+/-),SP-C(-/-)) mice failed to stabilize the surface tension of microbubbles, showing that SP-C plays a role in stabilization or recruitment of phospholipid films at low bubble radius. Genetically decreased levels of SP-B combined with superimposed O(2)-induced injury reveals the distinct contribution of SP-C to pulmonary function in vivo.

Synthesis and post-translational processing of surfactant protein C.

Traditional thinking about surfactant proteins has centered around their effects on the biophysical properties of surfactant phospholipids. Accumulated data now suggests that the four major surfactant proteins (SPs) are a biochemically and functionally diverse group of mammalian peptides that have function beyond modification of alveolar surface tension. Alveolar SP-C (SP-C3.7, Mr 21,000) is 35 amino acid peptide isolated from lung surfactant that is synthesized and processed from a 191-197 amino acid precursor (proSP-C21). Although its solubility in organic solvents and avidity for lipid membranes impart properties important for its biophysical activity, SP-C represents a structurally and functionally challenging protein for the alveolar type II cell that must synthesize and traffic the peptide through the regulated secretory pathway. Despite technical and analytical difficulties imposed by its unique structure, our current understanding of SP-C biosynthesis has evolved over the past 10 years. Recent data now require us to consider proSP-C21 as a hybrid molecule incorporating structural and functional features both of bitopic integral membrane proteins as well us more classically recognized propeptide hormones. Our article highlights major developments related to characterization of molecular and cellular mechanisms underlying expression, post-translational processing, and targeting of proSP-C21 that result in production of secreted SP-C3.7.

Restoring effects of vitamin A on surfactant synthesis in nitrofen-induced congenital diaphragmatic hernia in rats.

Congenital diaphragmatic hernia (CDH) is a major cause of refractory respiratory failure in the newborn. Besides pulmonary hypoplasia, the pathophysiology of CDH also includes surfactant deficiency. Vitamin A (vit A) is important for various aspects of lung development. We hypothesized that antenatal treatment with vit A would stimulate lung surfactant synthesis in experimental CDH induced in rats by maternal ingestion of the herbicide nitrofen (2,4-dichloro-phenyl-p-nitrophenyl-ether) on Day 12. Fetuses were assigned to six experimental groups: (1) controls from rats that received olive oil, the vehicle; (2) fetuses from rats that received olive oil on Day 12 and vit A orally (15,000 IU) on Day 14; (3) nitrofen (N)-exposed fetuses without diaphragmatic hernia (N/no DH); (4) N/no DH from rats given vit A on Day 14; (5 ) nitrofen-exposed fetuses with DH (N/+DH); (6) N/+DH from rats given vit A on Day 14. Fetuses were delivered by C-section at Day 21. Lung DNA content was lowered in the nitrofen group as compared with the controls group, but increased by subsequent vit A treatment. Lung surfactant disaturated phosphatidylcholine was reduced in the N/+DH group and restored to control level by vit A. The expression level of surfactant proteins (SP) -A and -C was decreased in vit A-treated control rats and in nitrofen-exposed fetuses with or without DH. Vit A restored SP-A and -C mRNA expression to control levels in N/+DH. SP-B expression was lowered in N/no DH and increased by vit A in this group. The proportion of type II cells assessed by SP-B immunolabeling was lowered in N/+DH and restored by vit A treatment. We conclude that antenatal treatment with vit A restores lung maturation in nitrofen-induced hypoplastic lungs with CDH. These findings point out vit A as a potential therapeutical agent for correcting surfactant deficiency in CDH.

Pulmonary alveolar proteinosis: a review.

Pulmonary alveolar proteinosis (PAP) is a disorder that rapidly leads to respiratory failure, because the alveolar spaces fill with a lipid-rich, proteinaceous material that impedes gas exchange. The pathogenesis of this life-threatening process remained an enigma for decades. Recent analysis of the lung pathology and molecular genetics of affected families has provided a molecular basis for some cases of PAP-deficiency of surfactant protein SP-B. This lack result from mutations in the gene for SP-B. The common mutation, 121ins2, is present in about two-third of the patients with SP-B deficiency. Additional insights into the mechanism for this lipoproteinaceous accumulation within alveoli were contributed by serendipity in a granulocyte-macrophage colony stimulating factor (GM-CSF) knock-out mouse model developed to study basal hematopoiesis. In this model, hematopoiesis was unaffected, but the animals developed pulmonary alveolar proteinosis. Subsequently, mutations in the genes for GM-CSF or its receptor were identified as the cause for pulmonary alveolar proteinosis in some patients. In our review, we discuss the known clinical, pathologic, and molecular genetic aspects of pediatric PAP and consider avenues for future research.

Neonatal high-frequency ventilation. Past, present, and future.

High-frequency ventilation has become established as an effective treatment modality in a variety of clinical situations. The laboratory and clinical investigations of these techniques have contributed tremendously to our understanding of the pathophysiology of respiratory failure and the important concept of maintaining adequate lung volume. Clinicians have come to appreciate better the factors involved in lung injury and the potential for damage to distant organs. The place of HFV in the therapeutic armamentarium will undoubtedly continue to evolve in the years to come. Of particular interest is the advent of advanced modes of fully synchronized and volume-targeted conventional mechanical ventilatory modes, along with the trend to use smaller tidal volumes and higher levels of PEEP with conventional ventilation. With these developments there seems to be a certain convergence of HFV and tidal ventilation that is the logical result of our improved understanding of respiratory pathophysiology. The available controlled trials of HFV versus tidal ventilation do not clearly differentiate whether improved outcomes are the result of HFV per se, or a reflection of the effects of optimizing lung volume, a benefit that may not be unique to HFV.

Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C.

Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4--7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.

Genetic disorders of neonatal respiratory function.

Genetic risk for respiratory distress in infancy has been recognized with increasing frequency in neonatal intensive care units. Reports of family clusters of affected infants and of ethnic- and gender-based respiratory phenotypes point to the contribution of inheritance. Similarly, different outcomes among gestationally matched infants with comparable exposures to oxygen, mechanical ventilation, or nutritional deficiency also suggest a genetic risk for respiratory distress. Examples of inherited deficiency of surfactant protein B in both humans and genetically engineered murine lineages illustrate the importance of identifying markers of genetic risk. In contrast to developmental, inflammatory, or nutritional causes of respiratory distress that may resolve as infants mature, genetic causes result in both acute and chronic (and potentially irreversible) respiratory failure. The availability of clinically useful genetic markers of risk for respiratory distress in infancy will permit development of rational strategies for treatment of genetic lung disorders of infancy and more accurate counseling of families whose infants are at genetic risk for development of respiratory distress at birth or during early childhood. We review examples of genetic variations known to be associated with or cause respiratory distress in infancy.

Surfactant protein deficiency in familial interstitial lung disease.

OBJECTIVE: To determine the contribution of surfactant protein abnormalities to the development of chronic lung injury in a familial form of interstitial lung disease. STUDY DESIGN: An 11-year-old girl, her sister, and their mother who were diagnosed with chronic interstitial lung disease underwent laboratory investigation of surfactant protein expression in bronchoalveolar lavage fluid and lung biopsy specimens. Nineteen patients with idiopathic pulmonary fibrosis and 9 patients who were investigated for pulmonary malignancy but who did not have interstitial lung disease served as control subjects. RESULTS: The 3 family members were found to have absent surfactant protein C (SP-C) and decreased levels of SP-A and SP-B in bronchoalveolar lavage fluid (BALF). Immunostaining for pulmonary surfactant proteins in lung biopsy specimens obtained from both children demonstrated a marked decrease of pro-SP-C in the alveolar epithelial cells but strong staining for pro-SP-B, SP-B, SP-A, and SP-D. No deviations from published surfactant protein B or C coding sequences were identified by DNA sequence analysis. All control subjects had a detectable level of SP-C in the BALF. CONCLUSION: The apparent absence of SP-C and a decrease in the levels of SP-A and SP-B are associated with familial interstitial lung disease.

Persistent improvement of gas exchange and lung mechanics by aerosolized perfluorocarbon.

The effect of aerosolized perfluorocarbon (PFC) (FC77) on pulmonary gas exchange and lung mechanics was studied in a surfactant depleted piglet model. Sixty minutes after induction of lung injury by bronchoalveolar lavage, 20 piglets were randomized to receive aerosolized PFC (Aerosol-PFC, 10 ml/kg/h, n = 5), partial liquid ventilation (PLV) at FRC capacity volume (FRC-PLV, 30 ml/kg, n = 5) or low volume (LV-PLV, 10 ml/kg/h, n = 5), or intermittent mandatory ventilation (IMV) (Control, n = 5). After 2 h, perfluorocarbon application was stopped and IMV was continued for 6 h. Sixty minutes after the onset of therapy, PaO2 was significantly higher and PaCO2 was significantly lower in the Aerosol-PFC and the FRC-PLV groups than in the LV-PLV and the Control groups; p < 0.001. Six hours after treatment, maximum PaO2 was found in the Aerosol-PFC group: 406.4 +/- 26.9 mm Hg, FRC-PLV: 217.3 +/- 50.5 mm Hg, LV-PLV: 96.3 +/- 18.9 mm Hg, Control: 67.6 +/- 8.4 mm Hg; p < 0.001. PaCO2 was lowest in the Aerosol-PFC group: 24.2 +/- 1.7 mm Hg, FRC-PLV: 35.9 +/- 2.8 mm Hg, LV-PLV: 56.7 +/- 12.4 mm Hg, Control: 60.6 +/- 5.1 mm Hg; p < 0.01. Dynamic compliance (C20/c) was highest in the Aerosol-PFC group; p < 0.01. Aerosolized perfluorocarbon improved pulmonary gas exchange and lung mechanics as effectively as PLV did in surfactant-depleted piglets, and the improvement was sustained longer.

Preserved spontaneous breathing improves cardiac output during partial liquid ventilation.

The aim of this study was to examine whether preserved spontaneous breathing (SB) supported by proportional-assist ventilation (PAV) would improve cardiac output (CO) during partial liquid ventilation (PLV) in rabbits with and without lung disease if compared with time-cycled, volume-controlled ventilation (CV) combined with muscle paralysis (MP). PLV was initiated in 17 healthy rabbits and 17 surfactant-depleted rabbits using 12 to 15 ml/kg of perfluorodecaline. Both ventilatory modes, SB+PAV and CV+MP, were applied in random sequence using a crossover design. CO was measured by thermodilution. CO was significantly higher during SB+PAV than during CV+MP: 136 +/- 21 ml/kg x min (mean +/- SD) versus 120 +/- 30 ml/kg x min (p = 0.004) in healthy rabbits, and 147 +/- 19 ml/kg x min versus 111 +/- 13 ml/kg x min (p < 0.0001) in surfactant-depleted rabbits, resulting in an improved oxygen delivery. This difference was mainly caused by a larger stroke volume during SB+PAV, whereas there was little change in heart rate. In surfactant-depleted rabbits, SB+PAV resulted in improved arterial blood pressure and arterial and mixed venous pH and in a higher PaO2 at the same level of PEEP and mean airway pressure. We conclude that during PLV, CO is higher during SB+PAV than during CV+MP, resulting in an improved oxygen delivery. In surfactant-depleted rabbits, improved CO, oxygen delivery, and arterial blood pressure resulted in higher pH, possibly reflecting improved tissue perfusion and oxygenation.

Partial liquid ventilation in severely surfactant-depleted, spontaneously breathing rabbits supported by proportional assist ventilation.

OBJECTIVE: We hypothesized that partial liquid ventilation (PLV) would improve oxygenation in nonparalyzed, surfactant-deficient rabbits breathing spontaneously while supported by proportional assist ventilation (PAV). This ventilation mode compensates for low pulmonary compliance and high resistance and thereby facilitates spontaneous breathing. DESIGN: Randomized trial. SETTING: University animal research facility. SUBJECTS: Twenty-six anesthetized New Zealand white rabbits weighing 2592 +/- 237g (mean +/- sd). INTERVENTIONS: After pulmonary lavage (target Pao2 <100 mm Hg on mechanical ventilation with 6 cm H2O of positive end-expiratory pressure [PEEP] and an Fio2 of 1.0), rabbits were randomized to PAV (PEEP of 8 cm H2O) with or without PLV. PLV rabbits received 25 mL/kg of perfluorocarbon by intratracheal infusion (1 mL/kg/min). Pao2, Paco2, tidal volume, respiratory rate, minute ventilation, mean airway pressure, arterial blood pressure, heart rate, pulmonary compliance, and airway resistance were measured. Evaporated perfluorocarbon was refilled every 30 mins in PLV animals. After 5 hrs, animals were killed and lungs were removed. Lung injury was evaluated using a histologic score. MAIN RESULTS: Pao2 and compliance were significantly higher in PLV rabbits compared with controls (p <.05, analysis of variance for repeated measures). All other parameters were similar in both groups. CONCLUSIONS: PLV improved oxygenation and pulmonary compliance in spontaneously breathing, severely surfactant-depleted rabbits supported by PAV. The severity of lung injury by histology was unaffected.

Surfactant protein D regulates NF-kappa B and matrix metalloproteinase production in alveolar macrophages via oxidant-sensitive pathways.

Targeted ablation of the surfactant protein D (SP-D) gene caused progressive pulmonary emphysema associated with pulmonary infiltration by foamy alveolar macrophages (AMs), increased hydrogen peroxide production, and matrix metalloproteinase (MMP)-2, -9, and -12 expression. In the present study, the mechanisms by which SP-D influences macrophage MMP activity were assessed in AMs from SP-D(-/-) mice. Tissue lipid peroxides and reactive carbonyls were increased in lungs of SP-D(-/-) mice, indicating oxidative stress. Immunohistochemical staining of AMs from SP-D(-/-) mice demonstrated that NF-kappaB was highly expressed and translocated to the nucleus. Increased NF-kappaB binding was detected by EMSA in nuclear extracts of AMs isolated from SP-D(-/-) mice. Antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited MMP production by AMs from SP-D(-/-) mice. To assess whether increased oxidant production influenced NF-kappaB activation and production of MMP-2 and -9, AMs from SP-D(-/-) mice were treated with the NADPH oxidase inhibitors diphenylene iodonium chloride and apocynin. Inhibition of NADPH oxidase suppressed NF-kappaB binding by nuclear extracts and decreased production of MMP-2 and 9 in AMs from SP-D(-/-) mice. SN-50, a synthetic NF-kappaB-inhibitory peptide, decreased MMP production by AMs from SP-D(-/-) mice. Oxidant production and reactive oxygen species were increased in lungs of SP-D(-/-) mice, in turn activating NF-kappaB and MMP expression. SP-D plays an unexpected inhibitory role in the regulation of NF-kappaB in AMs.