Surfactant Protein B Deficiency Research Articles

Allelic heterogeneity in hereditary surfactant protein B (SP-B) deficiency.

Inability to produce surfactant protein B (SP-B) causes fatal neonatal respiratory disease. A frame-shift mutation (121ins2) is the predominant but not exclusive cause of disease. To determine the range of mechanisms responsible for SP-B deficiency, both alleles from 32 affected infants were characterized. Sixteen infants were homozygous for the 121ins2 mutation, 10 infants were heterozygous for the 121ins2 and another mutation, and six infants were homozygous for other mutations. Thirteen novel SP-B gene mutations were identified, which were not found in a control population. One novel mutation was found in two unrelated families. Surfactant protein expression was evaluated by immunohistochemistry and/or protein blotting. Absence of proSP-B and mature SP-B was associated with nonsense and frame-shift mutations. In contrast, proSP-B expression was associated with missense mutations, or mutations causing in-frame deletions or insertions, and low levels of mature SP-B expression were associated with four mutations. Extracellular staining for proSP-C and/or aberrantly processed SP-C was observed in lungs of all infants with SP-B gene mutations. Hereditary SP-B deficiency is caused by a variety of distinct mutations in the SP-B gene and may be associated with reduced, as well as absent, levels of mature SP-B, likely caused by impaired processing of proSP-B.

RECURRENT FAMILIAL NEONATAL DEATHS: HEREDITARY SURFACTANT PROTEIN B DEFICIENCY

Hereditary surfactant protein B (SP-B) deficiency is an uncommon autosomal recessive lung disorder that causes hypoxemic respiratory failure in mature, morphologically normal infants. Recognition and diagnosis of this condition is of paramount importance, as it has significant implications for future pregnancies with a recurrence risk of 25%. In a family with three neonatal deaths over 20 years, SP-B deficiency was diagnosed following the death of the fourth affected infant. Previous deaths were mistakenly attributed to hyaline membrane disease (HMD), congenital Mycoplasma hominis infection, and pulmonary hypertension, however, following the diagnosis in the proposita, SP-B deficiency was also confirmed in her deceased siblings by immunohistochemical staining of autopsy specimens. This case highlights the presentation, postnatal course, diagnosis, and therapeutic options of SP-B deficiency in addition to the mode of inheritance and the possibility of antenatal diagnosis. Genetic consultation is imperative in the investigations of recurrent neonatal deaths, especially in cases of remote events. The recent enormous advances in human genetics have shown that many conditions previously ascribed to environmental agents have a genetic basis.

Surfactant protein-B-deficient mice are susceptible to hyperoxic lung injury.

Surfactant protein-B (SP-B) is a small, hydrophobic peptide that plays a critical role in pulmonary function and surfactant homeostasis. To determine whether SP-B protects mice from oxygen-induced injury, heterozygous SP-B(+/-) gene-targeted mice and wild-type SP-B(+/+) littermates were exposed to hyperoxia (95% oxygen for 3 d) or room air. Although specific lung compliance in room air in SP-B(+/-) mice was slightly reduced as compared with that in SP-B(+/+) mice, it was reduced more markedly during hyperoxia (46% versus 25% decrease, respectively). The larger decrease in lung compliance in SP-B(+/-) mice was associated with increased severity of pulmonary edema, hemorrhage and inflammation, lung permeability and protein leakage into the alveolar space. Hyperoxia increased SP-B messenger RNA (mRNA) and total protein concentrations by 2-fold in SP-B(+/+) and SP-B(+/-) mice, but decreased the abundance of SP-B protein in lavage fluid relative to total protein only in SP-B(+/-) mice. Hyperoxia increased SP-B expression, but apparently not enough to maintain SP-B function and lung compliance in the presence of increased protein leakage in SP-B(+/-) mice. Increased alveolar-capillary leakage and relative deficiency of SP-B may therefore contribute to oxygen-induced pulmonary dysfunction in SP-B(+/-) mice. These data support the concept that SP-B plays an important protective role in the lung.

Pulmonary surfactant in health and human lung diseases: state of the art.

Pulmonary surfactant is a complex and highly surface active material composed of lipids and proteins which is found in the fluid lining the alveolar surface of the lungs. Surfactant prevents alveolar collapse at low lung volume, and preserves bronchiolar patency during normal and forced respiration (biophysical functions). In addition, it is involved in the protection of the lungs from injuries and infections caused by inhaled particles and micro-organisms (immunological, non-biophysical functions). Pulmonary surfactant can only be harvested by lavage procedures, which may disrupt its pre-existing biophysical and biochemical micro-organization. These limitations must always be considered when interpreting ex vivo studies of pulmonary surfactant. A pathophysiological role for surfactant was first appreciated in premature infants with respiratory distress syndrome and hyaline membrane disease, a condition which is nowadays routinely treated with exogenous surfactant replacement. Biochemical surfactant abnormalities of varying degrees have been described in obstructive lung diseases (asthma, bronchiolitis, chronic obstructive pulmonary disease, and following lung transplantation), infectious and suppurative lung diseases (cystic fibrosis, pneumonia, and human immunodeficiency virus), adult respiratory distress syndrome, pulmonary oedema, other diseases specific to infants (chronic lung disease of prematurity, and surfactant protein-B deficiency), interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis), pulmonary alveolar proteinosis, following cardiopulmonary bypass, and in smokers. For some pulmonary conditions surfactant replacement therapy is on the horizon, but for the majority much more needs to be learnt about the pathophysiological role the observed surfactant abnormalities may have.

Use of Knockout Mice to Study Surfactant Protein Structure and Function

Pulmonary surfactant protein B (SP-B) is a 79 amino acid peptide that is intimately associated with surfactant phospholipids in the alveolar airspace. Mutations of the SP-B gene that result in complete absence of SP-B are invariably fatal in the neonatal period. The pathology associated with SP-B deficiency suggests that SP-B plays a critical role in integrating the synthesis, assembly and metabolism of the surfactant complex. A strategy is described to elucidate the role of SP-B in surfactant homeostasis by characterizing the pathophysiology associated with cell specific expression of SP-B constructs in vivo. Human SP-B constructs, under control of lung cell-specific promoters, were expressed in SP-B knockout mice in order to achieve expression of the human transgene in a null background. The effect of transgene expression on lung structure and function was assessed by biochemical, morphological and physiological analyses of the surfactant system in fetal and postnatal offspring.

Inhibition of lung surfactant protein B expression during Pneumocystis carinii pneumonia in mice

The pathogenesis of Pneumocystis carinii pneumonia (PCP) suggests an important role for dysfunction of the pulmonary surfactant system in the hypoxemic respiratory insufficiency associated with this infection. Surfactant protein B (SP-B) is a hydrophobic protein shown to be essential for normal surfactant function in vivo. Therefore, we hypothesized that the inhibition of SP-B expression occurs during PCP, and we tested this hypothesis in two immunodeficient animal models. PCP was induced in C.B-17 scid/scid mice by intratracheal inoculation of P. carinii organisms. Infected lung homogenates, obtained at time points up to 6 weeks after inoculation, were analyzed for SP-B and mRNA content. When a comparison was made with uninfected scid controls, the densitometric quantitation of Western blots of lung homogenates demonstrated significant reductions in 8 kd SP-B in mice infected with P. carinii 4 weeks after inoculation (16% of the control value). Northern blot analysis showed a concomitant decrease in SP-B mRNA to 24% of the control level. The decrease in SP-B and mRNA levels in lung homogenates of infected mice was reflected in lower SP-B levels in the surfactant. An enzyme-linked immunosorbent assay for the SP-B level in surfactant prepared from bronchoalveolar lavage samples of infected scid mice demonstrated a significant reduction in alveolar SP-B content (45% of the control value). In contrast to the results with SP-B, neither the SP-A protein content nor the mRNA level was significantly altered by PCP infection. To confirm these observations, SP-B expression was studied in an additional animal model of PCP. The SP-B content of lung homogenates from BALB/c mice depleted of CD4+ T cells and infected with P. carinii was also reduced (51% of the control value). We conclude that P. carinii induces selective inhibition of the expression of SP-B in two mouse models of PCP and that this down-regulation is mediated at the level of mRNA expression. Therefore, an acquired deficiency of SP-B is likely to be an important contributor to the pathogenesis of hypoxemic respiratory failure that is observed in patients with PCP.

Surfactant Phosphatidylcholine Metabolism in Patients with Congenital Surfactant Protein-B Deficiency Studied with Stable Isotopes

Surfactant Phosphatidylcholine Metabolism in Patients with Congenital Surfactant Protein-B Deficiency Studied with Stable Isotopes

Function and Inhibition Sensitivity of a Synthetic SP-B Mutant (R236C) in Preterm Rabbits

Presented in: Poster Session Monday, May 4, 1998, Poster number 195 † 1658 A point mutation in exon 7 of the human surfactant protein B (SP-B) gene, resulting in a cysteine for agrinine substitution (R236C), is associated with partial SP-B deficiency (Ballard et al., Pediatrics 1995). We synthesized, purified, and characterized this mutant SP-B (BR2326C), full length oxidized SP-B1-78(B), and palmitoylated SP-C1-35(C).

Regulation and Function of Pulmonary Surfactant Protein B

Pulmonary surfactant, a complex mixture of phospholipids and specific associated proteins, reduces the surface tension at the air-liquid interface of the distal conducting airways and gas exchanging alveoli of the lung. Lipids, primarily neutral and phospholipids, compose approximately 90% of the surfactant complex. The remaining 10% of surfactant is composed of at least three surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, and SP-C. These proteins contribute to the formation, stabilization, and function of organized surfactant structures. This article briefly reviews the normal composition and function of pulmonary surfactant and specifically reviews the structure, function, and regulation of surfactant protein B (SP-B). The recent identification of neonates with refractory respiratory failure due to a genetic absence of SP-B and the study of transgenic mice in which SP-B gene expression has been ablated highlight the importance of the protein to surfactant function, synthesis, and metabolism and to the maintenance of lung function. Gene reconstitution experimentsin vitroand in SP-B-deficient transgenic mice suggest specific functions for the amino and carboxyl terminal domains of the protein. SP-B deficiency is a potential target for gene therapy in human patients.

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Pulmonary surfactant, a complex mixture of phospholipids and specific associated proteins, reduces the surface tension at the air-liquid interface of the distal conducting airways and gas exchanging alveoli of the lung. Lipids, primarily neutral and phospholipids, compose approximately 90% of the surfactant complex. The remaining 10% of surfactant is composed of at least three surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, and SP-C. These proteins contribute to the formation, stabilization, and function of organized surfactant structures. This article briefly reviews the normal composition and function of pulmonary surfactant and specifically reviews the structure, function, and regulation of surfactant protein B (SP-B). The recent identification of neonates with refractory respiratory failure due to a genetic absence of SP-B and the study of transgenic mice in which SP-B gene expression has been ablated highlight the importance of the protein to surfactant function, synthesis, and metabolism and to the maintenance of lung function. Gene reconstitution experimentsin vitroand in SP-B-deficient transgenic mice suggest specific functions for the amino and carboxyl terminal domains of the protein. SP-B deficiency is a potential target for gene therapy in human patients.

Transient surfactant protein B deficiency in a term infant with severe respiratory failure

A 38-day-old male infant with persistent pulmonary hypertension and respiratory failure since birth was found to have a complete absence of surfactant protein B (SP-B) along with an aberrant form of SP-C in his tracheal aspirate fluid, findings consistent with the diagnosis of hereditary SP-B deficiency. Surprisingly, SP-B and SP-B messenger ribonucleic acid were present in lung biopsy tissue. However, DNA sequence analysis demonstrated a point mutation in exon 5 of one of the SP-B gene alleles. The infant's mother was found to be a carrier of this mutation. The infant's other SP-B allele did not differ from the published DNA sequence for the SP-B gene. We conclude that this patient had a transient deficiency of SP-B, in contrast to that of previously described infants with irreversible respiratory failure caused by hereditary SP-B deficiency. We recommend that infants with suspected SP-B deficiency have serial analysis of tracheal fluid samples for both SP-B and SP-C before lung biopsy, along with genetic analysis for the known SP-B mutations. We speculate that the new mutation found in one of this patient's SP-B genes was in part responsible for the transient deficiency of SP-B. (J Pediatr 1998;132:244-8)

Targeting type II and Clara cells for adenovirus-mediated gene transfer using the surfactant protein B promoter.

We examined the ability of the human surfactant protein B (SP-B) promoter to confer cell specificity of transgene expression in an adenoviral vector. Using similar replication-deficient adenoviruses (rAd), we compared lacZ reporter gene expression driven by the human SP-B promoter (rAd.SPBlacZ) with the ubiquitously expressed Rous sarcoma virus promoter (rAd.RSVlacZ). rAd.SPBlacZ expressed lacZ in H-441 and A549 lung epithelial cell lines and not in HeLa cells whereas rAd.RSVlacZ expressed in all three cell lines. In primary human fetal lung fibroblasts, beta-galactosidase activity from rAd.RSVlacZ transduction increased in a dose-dependent manner whereas activity from rAd.SPBlacZ remained low. In mixed cell cultures prepared from human fetal lung explants that contained fibroblasts and type II cells, X-Gal staining localized rAd.SPBlacZ expression to only type II cells whereas rAd.RSVlacZ expressed in both cell types. In 24-wk gestation human fetal tissue explants infected ex vivo, the RSV promoter directed lacZ expression in lung, trachea, heart, liver, and esophagus, whereas with the SP-B promoter lacZ was expressed only in lung, specifically in air space-lining cells. This specificity was maintained in vivo. lacZ expression was undetectable in lung and other tissues after intravenous administration of rAd.SPBlacZ whereas rAd.RSV-lacZ expressed primarily in liver. After intratracheal instillation of rAd.SPBlacZ into mice, X-Gal staining localized expression to type II and Clara cells. In contrast, rAd.RSVlacZ expressed in all pulmonary epithelial cell types. Our results indicate that the SP-B promoter may be useful in targeting type II and Clara cells for gene therapy of conditions such as inherited deficiency of SP-B.

Lung transplantation for treatment of infants with surfactant protein B deficiency

Objective: To evaluate lung transplantation for treatment of surfactant protein B (SP-B) deficiency. Study design: We compared surfactant composition and function from pretransplantation and posttransplantation samples of bronchoalveolar lavage fluid, somatic and lung growth, neurodevelopmental progress, pulmonary function, and pulmonary immunohistology in 3 infants with SP-B deficiency who underwent bilateral lung transplantation at 2 months of age and 3 infants who underwent lung transplantation for other reasons. Results: Two years after transplantation, the 2 surviving infants with SP-B deficiency exhibited comparable somatic growth and cognitive development to the comparison infants. All infants had delays in gross motor development that improved with time. Both groups have exhibited normal gas exchange, lung growth, and pulmonary function. The SP-B–deficient infants have also exhibited normal SP-B expression and pulmonary surfactant function after lung transplantation. In two SP-B–deficient infants antibody to SP-B developed. No pathologic consequences of this antibody were identified. Conclusions: Apart from the development of anti-SP-B antibody, the outcomes for SP-B–deficient infants after lung transplantation are similar to those of infants who undergo lung transplantation for other reasons. Lung transplantation offers a successful interim therapy until gene replacement for this disease is available. (J Pediatr 1997;130:231-9)

RETINOIC ACID AND GLUCOCORTICOIDS REGULATE SURFACTANT PROTEIN B GENE EXPRESSION IN HUMAN LUNG EPITHELIAL CELLS. • 337

Surfactant protein B (SP-B) plays a vital role in the pulmonary function of neonates; infants with inherited SP-B deficiency follow a course of progressive and fatal pulmonary failure unless they undergo lung transplantation. In previous studies, we have shown that all-trans retinoic acid (RA), a ligand of the steroid hormone receptor family, increases SP-B mRNA levels in H441 human lung adenocarcinoma cells and in cultured human fetal lung explants. In order to determine the temporal nature of the RA effect on SP-B mRNA, H441 cells were incubated with 10-6M all-trans RA for 1, 2, 4, 8, 12 or 24 hours. The stimulatory effect of all-trans RA on SP-B mRNA levels was observed as early as after 4 hours of exposure to the hormone. We also found that other retinoids, including 9-cis retinoic acid, 13-cis retinoic acid and retinol, do not affect SP-B mRNA levels in the H441 cell line. Glucocorticoids have been shown to increase SP-B mRNA levels in H441 cells and in cultured human fetal lung tissue. We hypothesized that the combination of glucocorticoids and all-trans RA regulates SP-B gene expression in a manner different from the effect of either agent alone. In initial experiments, H441 cells were treated in serum-free medium for 24 hours with cortisol (10-10M to 10-6M) added alone. SP-B mRNA levels were increased compared to control levels in a dose-dependent manner from 1.8 times over control levels in 10-9M cortisol to 4.7 times over control levels in 10-6M cortisol; there was no effect of 10-10M cortisol. H441 cells were then exposed to varying concentrations of cortisol (10-10 M to 10-6M) in the presence of all-trans RA (10-6M). SP-B mRNA levels were further stimulated to 2.8 times control levels in 10-9M cortisol and to 7 times control levels in 10-6M cortisol. At each concentration of cortisol, 10-9M to 10-6M, there was an average 40% increase in the stimulatory effect of cortisol on the level of SP-B mRNA with the addition of 10-6M all-trans RA. These data are suggestive that there is an additive effect of all-trans retinoic acid on cortisol-stimulated surfactant protein B gene expression in human lung epithelial cells.

Aberrant processing of surfactant protein C in hereditary SP-B deficiency.

Hereditary surfactant protein B (SP-B) deficiency causes lethal neonatal respiratory disease associated with abnormalities in pulmonary surfactant proteins and lipids. SP-C, a 4-kDa hydrophobic protein produced from a 21-kDa precursor, cooperates with SP-B to enhance the surface active properties of surfactant phospholipids. Anti-proSP-C polyclonal antisera were produced against fusion proteins containing 1) the amino terminus (amino acids 1-20), 2) the region carboxy-terminal to the mature SP-C peptide (amino acids 58-77), and 3) full-length 197-amino acid proSP-C and were characterized using immunoprecipitation, Western blot, and immunohistochemical techniques. Western blot analysis of bronchoalveolar lavage and amniotic fluid from hereditary SP-B-deficient patients allowed identification of a 12-kDa form of SP-C that contained epitopes consistent with the amino-terminal and active peptide regions of SP-C (amino acids 1-57). The 12-kDa SP-C peptide was not detected in bronchoalveolar lavage from healthy adults or adults with alveolar proteinosis or pneumonia. We conclude that SP-B deficiency is associated with the aberrant processing and secretion of an immature SP-C peptide, which may contribute to the respiratory failure associated with hereditary SP-B deficiency.

Pathophysiology and treatment of surfactant protein-B deficiency.

Surfactant protein B (SP-B) deficiency is an inherited disease of full-term newborn infants which leads to lethal respiratory failure within the first year of life. Genetic analysis of affected infants has permitted identification of a mutation in the SP-B gene found in several unrelated kindreds which disrupts pulmonary surfactant composition and function. Lung transplantation has resulted in reconstitution of pulmonary surfactant function and long-term survival. SP-B deficiency represents the first opportunity to link physiologic characteristics of respiratory failure in infancy with specific molecular and cellular defects. This linkage will facilitate development of novel strategies for the treatment of neonatal respiratory diseases.

Molecular and phenotypic variability in the congenital alveolar proteinosis syndrome associated with inherited surfactant protein B deficiency

Congenital alveolar proteinosis (CAP) is an often fatal cause of respiratory failure in term newborn infants, which has been associated with a genetic deficiency of surfactant protein B (SP-B) as a result of a frameshift mutation (121ins2) in a family with three affected siblings. In the index cases the deficiency of SP-B was associated with qualitative and quantitative abnormalities of the surfactant proteins A and C. Immunostaining for lung surfactant proteins and a search for the 121ins2 mutation by restriction enzyme analysis of DNA extracted from paraffin-embedded lung tissue was performed for 7 additional affected infants from 6 families, bringing to 10 the total number of patients with CAP who have been studied. In six infants, the surfactant protein immunostaining pattern was similar to that of the index cases. Of these, three patients were homozygous for the 121ins2 mutation; one was a compound heterozygote with the 121ins2 in one allele and a different mutation in the other; and three patients lacked the mutation in both alleles. One infant had an abundance of SP-B, suggesting phenotypic heterogeneity in CAP. Lung ultrastructural abnormalities, such as a reduced number of lamellar bodies, absent tubular myelin, and basal secretion of surfactant lipids and proteins, suggest a significant derangement of surfactant metabolism. The phenotypic heterogeneity in infants with CAP raises the possibility that variable degrees of SP-B deficiency may be more common than previously suspected. (J P EDIATR 1994;125:43-50)

A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds.

To determine the molecular defect accounting for the deficiency of pulmonary surfactant protein B (SP-B) in full-term neonates who died from respiratory failure associated with alveolar proteinosis, the sequence of the SP-B transcript in affected infants was ascertained. A frameshift mutation consisting of a substitution of GAA for C in codon 121 of the SP-B cDNA was identified. The three affected infants in the index family were homozygous for this mutation, which segregated in a fashion consistent with autosomal recessive inheritance of disease. The same mutation was found in two other unrelated infants who died from alveolar proteinosis, one of whom was also homozygous, and in the parents of an additional unrelated, affected infant, but was not observed in 50 control subjects. We conclude that this mutation is responsible for SP-B deficiency and neonatal alveolar proteinosis in multiple families and speculate that the disorder is more common than was recognized previously.