Surfactant protein D of the innate immune defence is inversely associated with human obesity and SP-D deficiency infers increased body weight in mice.

Surfactant protein D (SP-D) is a key regulator of pathogen-induced inflammation. SP-D is further involved in lipid homeostasis in mouse lung and circulation and recent data have demonstrated that the body mass index (BMI; in kg/m(2)) is influenced by genes in common with SP-D. The objective of the present study was to describe the association between serum SP-D and weight, waist circumference or BMI, and furthermore to observe body weight development in SP-D-deficient (Spd-/-) mice. As a part of the Danish population-based twin study (GEMINAKAR) on the metabolic syndrome, we analysed 1476 Danish twins for serum SP-D and investigated associations with weight, waist circumference and BMI by multiple regression analysis. Serum SP-D was significantly and inversely associated with weight (P = 0.001) and waist circumference in men (P < 0.001) and to BMI in both genders (P = 0.039 women, P < 0.001 men). The age-dependent increase in serum SP-D was most prominent in lean persons (BMI < 20). Spd-/- mice and wild-type mice were subjected to a feeding study and body weights were recorded in a time course over 24 weeks. Spd-/- mouse weight gain was significantly increased, with 90 mg/week (P < 0.0001) in males on normal chow. Fat percentage was significantly increased by 17% in the Spd-/- male mice (P = 0.003). We conclude, that there is an association between low levels or absent SP-D and obesity.

Early variable-flow nasal continuous positive airway pressure in infants < or =1000 grams at birth.

OBJECTIVE: To compare the incidence of chronic lung disease (CLD) in extremely low birth weight (ELBW, < or =1000 g) infants before and after the introduction of early, preferential application of nasal continuous airway pressure (NCPAP) utilizing a variable flow delivery system. STUDY DESIGN: A retrospective cohort study of ELBW infants 2 years prior to (Pre-early NCPAP, n=96) and 2 years following (Early NCPAP, n=75) the initiation of an early NCPAP policy. RESULTS: There were no significant changes (Pre-early NCPAP vs Early NCPAP) in the incidences of CLD (35 vs 33%, P=0.81) or CLD or death (50 vs 43%, P=0.34). Infants in the Early NCPAP group weaned off mechanical ventilation and supplemental oxygen more rapidly than infants in the Pre-early NCPAP group (hazard ratio (HR) 1.80, P=0.002 and HR 1.69, P=0.01). CONCLUSION: A policy of early NCPAP has not decreased the incidence of CLD despite a decrease in time to successful tracheal extubation.

The role of surfactant protein D in the colonisation of the respiratory tract and onset of bacteraemia during pneumococcal pneumonia.

We have shown previously that surfactant protein D (SP-D) binds and agglutinates Streptococcus pneumoniae in vitro. In this study, the role of SP-D in innate immunity against S. pneumoniae was investigated in vivo, by comparing the outcome of intranasal infection in surfactant protein D deficient (SP-D-/-) to wildtype mice (SP-D+/+). Deficiency of SP-D was associated with enhanced colonisation and infection of the upper and lower respiratory tract and earlier onset and longer persistence of bacteraemia. Recruitment of neutrophils to inflammatory sites in the lung was similar in both strains mice in the first 24 hrs post-infection, but different by 48 hrs. T cell influx was greatly enhanced in SP-D-/- mice as compared to SP-D+/+ mice. Our data provides evidence that SP-D has a significant role to play in the clearance of pneumococci during the early stages of infection in both pulmonary sites and blood.

Surfactant proteins in pulmonary alveolar proteinosis in adults.

Pulmonary alveolar proteinosis (PAP) is a rare disorder characterised histologically by an intra-alveolar accumulation of fine granular eosinophilic and periodic acid-Schiff positive material. In a retrospective study, the composition of the intra-alveolarly accumulated material of adult patients with PAP was analysed by means of immunohistochemistry and Western blotting. In patients with PAP, the current authors found an intra-alveolar accumulation of surfactant protein (SP)-A, precursors of SP-B, SP-B, variable amounts of mono-, di-, and oligomeric SP-C forms, as well as SP-D. Only in one patient was a precursor of SP-C detected. By means of immuno-electron microscopy, the current authors identified not only transport vesicles labelled for precursors of SP-B and SP-C, but also transport vesicles containing either precursors of SP-B or SP-C in type-II pneumocytes in normal human lungs. It is concluded that pulmonary alveolar proteinosis in adults is characterised by an intra-alveolar accumulation of surfactant protein A, precursors of surfactant protein B, and surfactant proteins B, C and D. The current data provide evidence that not only an impairment of surfactant clearance by alveolar macrophages, but also an abnormal secretion of transport vesicles containing precursors of surfactant protein B (but not surfactant protein C) and an insufficient palmitoylation of surfactant protein C, which may lead to the formation of di- and oligomeric surfactant protein C forms, play a role in the pathogenesis of pulmonary alveolar proteinosis.

Surfactant protein D deficiency influences allergic immune responses.

BACKGROUND: The collectin surfactant protein D (SP-D) confers protection against pulmonary infection and inflammation. Recent data suggest a role for SP-D in the modulation of allergic inflammation. OBJECTIVE: The aim of this study is to characterize the immune responses of SP-D-deficient (SP-D(-/-)) mice in a kinetic model of allergic inflammation. We determined whether allergic parameters were enhanced in SP-D(-/-) mice in vivo. Further, we examined whether functional immune responses in vitro such as lymphocyte proliferation (LP) and cytokine production were modulated in the absence of SP-D. METHODS: In vivo, wild-type (WT) and SP-D(-/-) mice were sensitized and challenged with the allergen ovalbumin (OVA) and assessed for allergic parameters (bronchoalveolar lavage (BAL) eosinophils, IL-13 production, pulmonary IFN-gamma, IL-10 expression) at early time points (1 and 3 days of challenge) in comparison with late time points (7 days of challenge). In vitro, spleen cells from WT and SP-D(-/-) mice were stimulated with the mitogen concanavalin A (ConA) and lipid A (LpA) and analysed for LP, IL-13 and IFN-gamma production. Toll-like receptor 4 (TLR4), ligand for LpA, was assessed by mRNA expression and immunohistochemistry in vivo. RESULTS: Following allergen exposure in vivo, SP-D(-/-) mice expressed higher BAL eosinophils and IL-13 concentrations and lower IFN-gamma expression at early time points compared with WT mice. IL-10 expression was increased at early time points in SP-D(-/-) compared with WT mice. Allergen-induced TLR4 expression was increased in WT, but not in SP-D(-/-) mice. After stimulation with LpA and ConA in vitro LP was increased and IFN-gamma concentration was decreased in SP-D(-/-) mice. CONCLUSION: SP-D may be critical for the modulation of early stages of allergic inflammation in vivo.

Fetoscopic temporary tracheal occlusion by means of detachable balloon for congenital diaphragmatic hernia.

Occlusion of the fetal trachea blocks the egress of fetal lung fluid and stimulates the growth of hypoplastic lungs in fetuses with diaphragmatic hernia. Accomplishing temporary and reversible occlusion of the fetal trachea has proven difficult without invasive fetal surgery. Using simultaneous real-time ultrasonography and fetal bronchoscopy through a single uterine port, we placed a detachable balloon in the trachea of 2 fetuses with severe diaphragmatic hernia. In both fetuses the fetal lung subsequently enlarged, allowing survival after birth.

GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice.

Mice deficient in surfactant protein (SP) D develop increased surfactant pool sizes and dramatic changes in alveolar macrophages and type II cells. To test the hypothesis that granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates alveolar macrophage proliferation and activation and the type II cell hypertrophy seen in SP-D null mice, we bred SP-D and GM-CSF gene-targeted mice to obtain littermate double null, single null, and wild-type mice. Bronchoalveolar lavage levels of phospholipid, protein, SP-D, SP-A, and GM-CSF were measured from 1 to 4 mo. There was an approximately additive accumulation of phospholipid, total protein, and SP-A at each time point. Microscopy showed normal macrophage number and morphology in GM-CSF null mice, numerous giant foamy macrophages and hypertrophic type II cells in SP-D null mice, and large but not foamy macrophages and mostly normal type II cells in double null mice. These results suggest that the mechanisms underlying the alveolar surfactant accumulation in the SP-D-deficient and GM-CSF-deficient mice are different and that GM-CSF mediates some of the macrophage and type II cell changes seen in SP-D null mice.

The pulmonary collectins and surfactant metabolism.

Lung surfactant covers and stabilizes a large, delicate surface at the interface between the host and the environment. The surfactant system is placed at risk by a number of environmental challenges such as inflammation, infection, or oxidant stress, and perhaps not surprisingly, it demonstrates adaptive changes in metabolism in response to alterations in the alveolar microenvironment. Recent experiments have shown that certain components of the surfactant system are active participants in the regulation of the alveolar response to a wide variety of environmental challenges. These components are capable not only of maintaining a low interfacial surface tension but also of amplifying or dampening inflammatory responses. These observations suggest that regulatory molecules are capable of both sensing the environment of the alveolus and providing feedback to the cells regulating surfactant synthesis, secretion, alveolar conversion, and clearance. In this review we examine the evidence from in vitro systems and gene-targeted mice that two surfactant-associated collectins (SP-A and SP-D) may serve in these roles and help modify surfactant homeostasis as part of a coordinated host response to environmental challenges.

Inhaled nitric oxide in neonates with persistent pulmonary hypertension.

To investigate the oxygenation and haemodynamic dose response to inhaled nitric oxide in neonates with persistent pulmonary hypertension (PPHN), we gave seven neonates nitric oxide and measured directly pulmonary arterial pressure. Inhaled nitric oxide produced peak improvement in oxygenation at 5 parts per million (ppm) whereas peak improvement in the pulmonary-to-systemic arterial pressure ratio did not occur until a nitric oxide dose of 20 ppm, which suggests that an Initial dose of 20 ppm is optimum for the treatment of PPHN.

Characterization of the mouse collectin gene locus.

Three of the four known mouse collectin genes have been mapped to chromosome 14. To further characterize the spatial relationship of these genes, a bacterial artificial chromosome (BAC) library of mouse chromosome 14 was screened using mouse surfactant protein (SP)-A and -D complementary DNAs (cDNAs). One large clone hybridized to both SP-A and SP-D cDNAs and was found by polymerase chain reaction (PCR) to contain sequences from one of the mouse mannose-binding lectin genes (Mbl1). We used Southern mapping and subcloning of overlapping restriction fragments to characterize the gene locus. Mapping was confirmed by fluorescent in situ hybridization of fiber-stretched BAC DNA and by Southern hybridization of restriction endonuclease-digested and PCR-amplified genomic DNA. We found that the SP-A, Mbl1, and SP-D genes reside contiguously within a 55-kb region. The SP-A and Mbl1 genes are in the same 5' to 3' orientation and 16 kb apart. The SP-D gene is in the opposite orientation to the two other collectin genes, 13 kb away from the 3' end of the Mbl1 gene. The mouse SP-D gene had not previously been characterized. We found its size (13 kb) and organization to be similar to that of human SP-D. Exon I is untranslated. The second exon is a hybrid exon that contains signal for initiation of translation, signal peptide, N-terminal domain, and the first seven collagen triplets of the collagen-like domain of the protein. Four short exons (III through VI) encode the collagen-like domain of the protein, and exons VII and VIII the linking and the carbohydrate-recognition domains, respectively.

Ultrastructure of phospholipid mixtures reconstituted with surfactant proteins B and D.

Surfactant protein (SP)-D is secreted from pulmonary alveolar type II cells into the alveolar lumen where potential interactions with surfactant lipids might occur. SP-D binds phosphatidylinositol (PI), a component of mammalian surfactants that is increased in a variety of injury states. We investigated the ultrastructure and properties of lipid protein recombinants that included SP-D, PI, and SP-B and compared these with recombinants based on SP-A. SP-D had a profound effect on the organization of phospholipid vesicles containing PI and SP-B, promoting the formation of atypical but highly ordered and surface-active tubular aggregates distinct in their dimensions and shape from the classical tubular myelin formed by SP-A. We also found both types of tubules in the secretions of type II cells maintained in long-term culture. These results suggest that surface atypical tubules can be formed with SP-D in vitro and in vivo.

Localization of a candidate surfactant convertase to type II cells, macrophages, and surfactant subfractions.

Pulmonary surfactant exists in the alveolus in several distinct subtypes that differ in their morphology, composition, and surface activity. Experiments by others have implicated a serine hydrolase in the production of the inactive small vesicular subtype of surfactant (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222-230, 1990). Our laboratory recently identified this enzyme in the rat as the serine carboxylesterase ES-2 [F. Barr, H. Clark, and S. Hawgood. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L404-L410, 1998]. In the present study, we determined the cellular sites of expression of ES-2 in rat lung using a digoxygenin-labeled ES-2 riboprobe. ES-2 mRNA was localized to type II cells and alveolar macrophages but not to Clara cells. Using a specific ES-2 antibody, we determined the protein distribution of ES-2 in the lung by immunohistochemistry, and it was found to be consistent with the sites of mRNA expression. Most of the ES-2 in rat bronchoalveolar lavage is in the surfactant-depleted supernatant, but ES-2 was also consistently localized to the small vesicular surfactant subfraction presumed to form as a consequence of conversion activity. These results are consistent with a role for endogenous lung ES-2 in surfactant metabolism.

Structure and properties of surfactant protein B.

Surfactant protein B is a small homodimeric protein that is found tightly associated with surfactant lipids in the alveolar space. In this review, we discuss the actions of SP-B on phospholipid membranes using information predominantly obtained from model membrane systems. We try to correlate these model actions with current concepts of SP-B structure and proposed biological functions. These functions may include critical roles in the intracellular assembly of surfactant through a role in lamellar body organogenesis, the structural rearrangement of secreted surfactant lipids into tubular myelin, and the subsequent rapid insertion of secreted surfactant phospholipids into the surface film itself. The relevance of SP-B to human biology is emphasized by the fatal respiratory distress that is associated with a genetic deficiency of SP-B and the important role of SP-B in certain exogenous surfactant formulations in wide clinical use.

Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D.

Surfactant protein D (SP-D) is one of two collectins found in the pulmonary alveolus. On the basis of homology with other collectins, potential functions for SP-D include roles in innate immunity and surfactant metabolism. The SP-D gene was disrupted in embryonic stem cells by homologous recombination to generate mice deficient in SP-D. Mice heterozygous for the mutant SP-D allele had SP-D concentrations that were approximately 50% wild type but no other obvious phenotypic abnormality. Mice totally deficient in SP-D were healthy to 7 months but had a progressive accumulation of surfactant lipids, SP-A, and SP-B in the alveolar space. By 8 weeks the alveolar phospholipid pool was 8-fold higher than wild-type littermates. There was also a 10-fold accumulation of alveolar macrophages in the null mice, and many macrophages were both multinucleated and foamy in appearance. Type II cells in the null mice were hyperplastic and contained giant lamellar bodies. These alterations in surfactant homeostasis were not associated with detectable changes in surfactant surface activity, postnatal respiratory function, or survival. The findings in the SP-D-deficient mice suggest a role for SP-D in surfactant homeostasis.

Correction of congenital diaphragmatic hernia in utero IX: fetuses with poor prognosis (liver herniation and low lung-to-head ratio) can be saved by fetoscopic temporary tracheal occlusion.

BACKGROUND/PURPOSE: Fetuses with congenital diaphragmatic hernia (CDH) who have a "poor prognosis" with postnatal treatment now can be identified on the basis of liver herniation, early diagnosis (before 25 weeks' gestation) and a low lung-to-head ratio (LHR). Because complete in utero repair proved unsuccessful for this group, the strategy of temporary tracheal occlusion was developed to gradually enlarge the hypoplastic fetal lung. The purpose of this study is to compare the outcome of patients in the poor-prognosis group treated by one of three methods: (1) standard postnatal care, (2) fetal tracheal occlusion via open hysterotomy, and (3) the recently developed video-fetoscopic (Fetendo) technique of tracheal occlusion without hysterotomy. METHODS: In the past 3 years, 34 of 86 fetuses with an isolated left CDH met criteria for the poor-prognosis group. Thirteen families chose postnatal treatment at an extracorporeal membrane oxygenation (ECMO) center, 13 underwent open fetal tracheal occlusion, and eight underwent fetoscopic tracheal occlusion. RESULTS: The survival rate was 38% in the group treated by standard postnatal therapy, 15% in the open tracheal occlusion group, and 75% in the Fetendo group. There were less postoperative pulmonary complications noted in mothers who underwent the Fetendo procedure versus the open tracheal occlusion. All but one Fetendo clip patient had a striking physiological response demonstrated by sonographic enlargement of the small left lung that was documented postnatally by plain radiographs and its subjective appearance during repair of the CDH. In contrast, only 5 of the 13 open tracheal occlusion patients demonstrated lung growth. CONCLUSION: Fetuses with a left CDH who have liver herniation and a low LHR are at high risk of neonatal demise and appear to benefit from temporary tracheal occlusion when performed fetoscopically, but not when performed by open fetal surgery.

Identification of a novel alternatively spliced mRNA of murine pulmonary surfactant protein B.

An alternatively spliced mRNA of pulmonary surfactant protein B (SP-B) was identified in murine lung. Sequencing analysis revealed a 69 base-pair (bp) deletion at the beginning of exon 7 of SP-B, presumably the result of an alternative splicing event. Reverse transcription-polymerase chain reaction (RT-PCR) of mouse, rat, and rabbit lung RNA revealed the existence of full-length and the 69-bp deleted short form. Ribonuclease protection assay of the SP-B messenger RNA (mRNA) demonstrated expression of both isoforms in five strains of adult and fetal mice with different genetic backgrounds, as well as in rabbit, but not in human. Splice junction sequences in exon 6 and at the exon 7 splice boundary for the two isoforms are similar, including AG doublet identity, but sequence differences do not account for species variation in isoform abundance. The abundance of the short SP-B mRNA isoform was approximately 30% of total SP-B mRNA in mouse and rabbit. Analysis of precursor SP-B protein in mouse lung suggested that the two mRNA species are expressed as stable protein isoforms.

Identification of a putative surfactant convertase in rat lung as a secreted serine carboxylesterase.

In the alveolar lumen, pulmonary surfactant converts from the contents of secreted lamellar bodies to tubular myelin to apoprotein-depleted vesicles during respiration. Using an in vitro system, researchers have reported that the conversion of tubular myelin to vesicles is blocked by inhibitors of serine hydrolase activity and have tentatively ascribed "convertase" activity to a diisopropyl fluorophosphate (DFP)-binding protein in mouse bronchoalveolar lavage (BAL). We purified and sequenced the homologous enzyme from rat BAL fluid. Amino acid sequence from the amino terminus and an internal cyanogen bromide peptide of the purified rat DFP-binding protein perfectly match the sequence of the carboxylesterase ES-2. Although ES-2 was initially cloned from liver, we found a 1.8-kilobase mRNA for ES-2 in decreasing relative abundance in rat liver, kidney, and lung but not in heart or spleen. Although further studies are required to establish the identity between "convertase" and ES-2 or a homologous member of the carboxylesterase family, our results raise the possibility that a protein with esterase/lipase activity plays a role in extracellular surfactant metabolism.

Tracheal ligation increases cell proliferation but decreases surfactant protein in fetal murine lungs in vitro.

Tracheal occlusion affects both fetal lung growth and maturation. The authors used a murine in vitro whole organ culture model to investigate these effects. The authors hypothesized that tracheal ligation would increase lung growth by increasing cell proliferation and would change surfactant protein synthesis in this system. Lungs were removed from day 14 gestation murine fetuses (term, 21 days). Tracheas were ligated and explants cultured in chemically defined, serum-free media for 1, 3, 4, 5, 7, or 14 days. DNA synthesis and cell division were assessed using a 5-bromo-2'-deoxy-uridine (BrdU) incorporation assay. Surfactant proteins A and B, markers of lung maturity, were detected using immunohistochemistry. Ligated lungs showed more BrdU-labeled cells per 1,000 x field (cells/hpf) at every time point. Ligated lungs on day 1 showed 27% more cells/hpf than unligated, on day 3, 21% more, on day 5, 54% more, on day 7, 60% more, and on day 14, 123% more (P < .05). In contrast, ligated lungs showed significantly less staining for surfactant proteins A and B than did unligated lungs. The authors conclude that tracheal ligation increases cell division but decreases surfactant protein in fetal murine lungs in vitro. These data suggest that although tracheal occlusion increases lung growth, it may decrease or delay lung maturation. This model provides a powerful tool for investigating the mechanisms underlying fetal lung development and tracheal occlusion-induced pulmonary hyperplasia.

Effects of mechanical factors on growth and maturation of the lung in fetal sheep.

Previous fetal studies indicated that endocrine factors control surfactant maturation, whereas mechanical forces affect lung growth, but not surfactant. We altered mechanical forces in fetal sheep lungs at 100-108 days gestation by tracheal ligation (TL, n = 15, 7 successful studies) to accelerate lung growth, transection of cervical spinal cord (TCSC, n = 17, 6 successful studies) to produce lung hypoplasia, or sham operation (n = 11, 6 successful studies). The reasons for the high mortality rates are not known. At delivery (130-142 days), groups were similar in gestational age, weight, and cortisol. Effects on lung growth were similar to, but effects on surfactant differed from, previous reports. TL increased lung growth but decreased saturated phosphatidylcholine (SatPC) and surfactant protein (SP)A and apparently decreased SP-B and relative numbers of alveolar type II cells (based on immunohistochemical studies of 1 animal in each group); TCSC had opposite effects. In contrast to a previous study (J. A. Kitterman, G. C. Liggins, G. A. Campos, J. A. Clements, C. S. Forster, C. H. Lee, and R. K. Creasy, J. Appl. Physiol, 51: 384-390, 1981), SatPC did not correlate with cortisol. We conclude that altering mechanical forces in fetal lung affects not only lung growth but also surfactant maturation and possibly alveolar epithelial differentiation and disturbs the normal correlation between cortisol and surfactant. Associated changes in insulin-like growth factor I (IGF-I; increased by TL, P = 0.003) suggest a possible role for IGF-I in these effects.