Pulmonary surfactant isolated by lung lavage consists of highly heterogeneous forms of phospholipid–protein aggregates of distinct sizes, structural characteristics, and composition. Tubular myelin is the most abundant form of alveolar phospholipid and consists of large, relatively dense aggregates (termed large aggregate surfactant) composed of phospholipids and surfactant proteins (SPs). Tubular myelin is a highly organized form of surfactant phospholipid, forming square tubular arrays. Tubular myelin represents an extracellular pool of surfactant lipids that rapidly moves to the air–liquid interface and reorganizes to form multilayered sheets that reduce surface tension in the alveolus (Fig. 5-2). Large lamellated structures, with lipid composition similar to that of tubular myelin, are seen within the alveolus and likely represent newly secreted lamellar bodies that unravel to form tubular myelin in the alveoli. The phospholipid composition of lamellar bodies, the intracellular storage form of surfactant, tubular myelin, and lamellated forms present in the alveolus are virtually identical. Smaller, less dense particles (small aggregate surfactant) are also present within the alveolar space, representing remnants or catabolic forms of surfactant that have relatively poor surface activity. Small aggregate surfactant is destined for uptake, reutilization, or catabolism by type II epithelial cells and catabolism by alveolar macrophages.2,3
Surfactant Phospholipids and Proteins
The composition of surfactant lipids is similar in all of the structural forms of surfactant isolated from mammalian lungs, with phospholipids generally representing 80% to 90% of the mass of pulmonary surfactant.3 In the adult lung, phosphatidylcholine (PC) and phosphatidylglycerol (PG) are the most abundant phospholipids, representing approximately 70% to 80% and 5% to 10%, respectively, of the lipid mass. Dipalmitoyl phosphatidylcholine (DPPC) is the most abundant species of PC. Lesser amounts of phosphatidylserine, phosphatidylethanolamine, sphingomyelin, neutral lipids (mostly cholesterol), and glycolipids are also present in surfactant. The lung content of surfactant phospholipids increases markedly with advancing gestation, regulated by a complex signaling and transcriptional network that controls type II alveolar cell differentiation, lipid synthesis, and SP gene expression. Lamellar bodies are secreted into the fetal amniotic fluid. PC, lamellar body counts, DPPC content, and increased lecithin (PC) to sphingomyelin (L/S) ratio, correlate with postnatal respiratory function. These tests are used to predict pulmonary maturity prior to the birth of preterm infants. Lung maturation and synthesis of surfactant components are induced by maternal administration of glucocorticoids, used clinically to prevent respiratory distress prior to premature birth.7 Proteins represent approximately 5% to 15% of the mass of pulmonary surfactant and include serum proteins and proteins that are synthesized and secreted by type II alveolar epithelial cells. In addition to its specific interaction with SP-B, the anionic phospholipid PG may also play an important role in innate defense. PG constitutes 10 mole% of surfactant lipid, with palmitoyl-oleoyl-PG (PoPG) being the most common species in human surfactant. PoPG specifically suppresses LPS-induced inflammatory responses and prevents infection of epithelial cells by binding RSV or influenza A virus.8 Thus the unique enrichment of PoPG in the distal airspaces may be an important component of host defense against inhaled pathogens. Four surfactant proteins (SPs), SP-A, SP-B, SP-C, and SP-D, are produced by respiratory epithelial cells, each playing specific roles in surfactant homeostasis and innate host defense.2,3,9,10
Surfactant is uniquely enriched in disaturated DPPC. The saturated C16 acyl chains pack densely at an air–liquid interface, reducing tension at the surface. However, dense and stable packing of DPPC occurs at a phase transition of 41°C, far above physiologic temperatures. Thus, at 37°C, pure DPPC maintains a semicrystalline or gel phase that is incapable of moving rapidly with the expansion and compression of the alveoli during the respiratory cycle. The capability of DPPC pulmonary surfactant to move rapidly to the alveolar interface at 37°C and to maintain low surface tension during dynamic compression is conferred by the surfactant-associated proteins SP-B and SP-C. PC synthesis in the lung is controlled by genes encoding choline phosphate cytidylyltransferase (PCYTLA) and choline kinase (CHKA), which are required for surfactant lipid synthesis and lung function at birth. DPPC is synthesized in type II alveolar cells via both a de novo pathway and remodeling of lysoPC. The enzyme lysoPC acetyltransferase (LPCAT1) mediates reacylation during surfactant lipid biosynthesis. Surfactant lipids, synthesized in the endoplasmic reticulum (ER) are transferred via a Golgi-independent pathway to lamellar bodies, the major intracellular storage site of surfactant (Fig. 5-2). Transfer of lipids occurs via nonvesicular transport and uptake into lamellar bodies requires the ABCA3 transporter, which selectively transports PC and PG. In contrast, surfactant proteins SP-B and SP-C traffic from the ER to the Golgi and subsequently to multivesicular bodies where proteolytic processing is initiated. Ultimately, multivesicular bodies are incorporated into lamellar bodies with surfactant lipids prior to secretion from type II alveolar cells.3
Structure and Function of Surfactant Proteins
Four distinct surfactant-associated proteins have been isolated from surfactant obtained by lung lavage. Their cDNAs, genes, and structures have been identified and are well characterized (Table 5-1).9,10 The SPs are expressed in a relatively lung epithelial cell–selective manner and are secreted into the airspaces, where they influence the structure, metabolism, and function of surfactant. Two classes of proteins have been distinguished on the basis of their structures. SP-A and SP-D are relatively abundant, hydrophilic, structurally related proteins and are members of the calcium-dependent lectin family of proteins that have similar amino-terminal collagenous and C-terminal lectin domains.11 SP-A and SP-D have little “surfactant”-like qualities but are able to bind complex carbohydrates, lipids, and glycolipids, including those on the surface of cells, bacteria, viruses, fungi, and other lung pathogens. SP-A and SP-D influence the structural forms and metabolism of surfactant lipids in the alveolus. They act as opsonins, activate alveolar macrophages, and play important roles in innate host defense in the lung. In contrast, SP-B and SP-C are small, hydrophobic proteins that play critical roles in enhancing the rate of spreading and stability of surfactant phospholipids needed to optimally reduce surface tension.10 SP-B and SP-C are the sole protein components of the animal-derived surfactant replacement preparations used for the treatment of IRDS at present.10
Table 5-1Regulation of Surfactant Homeostasis |Favorite Table|Download (.pdf) Table 5-1Regulation of Surfactant Homeostasis
|Genes/locus ||Functions ||Inheritance ||Presentation ||Age at Presentation |
|Lipid transport |
|AR ||RDS |
|Surfactant packaging/function ||AR ||RDS ||Newborns |
|Surfactant function ||AD ||ILD > RDS ||Infants, children, adults |
|Tubular myelin, host defense ||AD ||ILD/lung cancer ||Adults |
|Lung, thyroid, CN morphogenesis, surfactant regulation ||AR |
|Alveolar macrophage function ||AR ||PAP ||Children |
|Alveolar macrophage function ||Autoimmune ||PAP ||Adults |
Surfactant Protein B (SP-B)
SP-B is a hydrophobic, amphipathic 8.8-kDa protein produced from a single gene (SFTPB, OMIM 178640) located on human chromosome 2. The SP-B mRNA is expressed in nonciliated bronchiolar cells and type II alveolar cells and is translated to produce a 40- to 42-kDa precursor that is proteolytically processed in the secretory pathway of type II epithelial cells to form the active 79-amino acid peptide found in alveolar surfactant. In combination with lipids, SP-B can reconstitute most of the surface activity of natural lung surfactant. SP-B contains two regions, (Trp9-Pro23) and (Ile56-Pro67), predicted to form amphipathic α-helices that interact with the surface of lipid films. Almost 50% of the protein is in an α-helical conformation; the amphipathic domains of SP-B interact with surfactant lipids, and PG in particular, to promote lipid incorporation into and stabilization of the surface film. SP-B contains three intramolecular disulfide bonds that confine the amphipathic helices of SP-B in an antiparallel configuration. Intermolecular disulfide bonds stabilize SP-B dimers. Dimers and higher multimers of SP-B, which are probably stabilized by noncovalent interactions, are readily identified in pulmonary surfactant.
ProSP-B is synthesized in the ER and proteolytically processed in multivesicular and lamellar bodies by cathepsins and other intracellular proteases. The active SP-B peptide is packaged with SP-C and surfactant lipids in lamellar bodies prior to secretion into the alveolus. In the alveoli, the positively charged amino acid residues of SP-B selectively interact with the negatively charged phospholipid DPPG. In a mixed DPPC/DPPG monolayer, SP-B is believed to purify the DPPC monolayer by removal of DPPG. SP-B increases order in the lipid head groups with little effect on order on acyl chains in the lipid membrane interior. The ability to order the lipid head group region is located in the amino- and carboxy-terminal regions of SP-B (1–20) and (53–78), which contain the predicted amphipathic helices. Synthetic peptides that contain these two regions have surface-tension–lowering activity similar to that of native SP-B and peptide mimics have been developed for therapy of respiratory distress in infants. SP-B enhances the insertion (fusion) of phospholipid vesicles into a preformed DPPC/DPPG monolayer, particularly in the presence of divalent cations. SP-B causes lipids in solution to form discoid particles often appearing as stacks or sheets. Together with SP-A, lipids, and Ca2+, SP-B reconstitutes the characteristic ultrastructural features of tubular myelin, producing multilamellar aggregates and square lattice configurations.
SP-B is Required for Survival After Birth
Mutations in the gene encoding SP-B (SFTPB, OMIM 265120) cause acute respiratory failure at birth related to surfactant dysfunction.12,13 Similarly, deletion of Sftpb in the developing lung or its conditional deletion in adult mice causes acute respiratory distress associated with alveolar capillary leak and surfactant deficiency.14 Pathologic findings of patients with lung disease related to SFTPB mutations are similar to those in mice in which the Sftpb gene is deleted.14,15 SP-B deficiency is associated with failure to form lamellar bodies, accumulation of abnormal multivesicular bodies within the type II cells, and failure to form tubular myelin or functional surfactant films after secretion into the alveoli.
Hereditary SP-B Deficiency Causes Respiratory Failure at Birth
SP-B deficiency is inherited in an autosomal recessive pattern, affected infants developing respiratory failure shortly after birth. While lung morphogenesis proceeds normally in utero, the lack of SP-B results in acute atelectasis and respiratory distress, usually presenting as full-term infants with signs and symptoms of diffuse alveolar collapse after birth. More than 40 distinct SFTPB mutations in patients with SP-B deficiency have been identified.13,15 The disorder is refractory to surfactant replacement therapy and most patients die from respiratory failure within several months after birth, requiring oxygen and ventilatory support throughout their clinical course. Lung transplantation has been offered to some patients. SP-B deficiency disrupts the formation of lamellar bodies and tubular myelin, and interferes with the processing of proSP-C to the active peptide. Thus, most SP-B–deficient patients lack both SP-B and SP-C peptides in the alveolus. In patients with SFTPB-related disease, proSP-C accumulates in the airspaces, contributing to an alveolar proteinosis–like syndrome. Pathologic diagnoses include desquamative interstitial pneumonitis (DIP), chronic pneumonitis of infancy (CPI) or infantile alveolar proteinosis, histologic changes being influenced by age, and supportive therapies (Fig. 5-3).16 The definitive diagnosis is made by identification of SFTPB gene mutations, enabling prenatal diagnosis and genetic counseling. While various missense, nonsense, frameshift, and splice variants have been identified, 121ins2 mutation in exon 4 is the most common mutation.12 In most affected infants, SP-B is lacking in bronchoalveolar lavage fluid and the abnormal proSP-C peptide accumulates in the alveoli, findings that can be verified by immunohistochemistry. Patients with SP-B deficiency do not respond to surfactant replacement and generally succumb from chronic respiratory failure early in infancy in spite of intensive care.
Pulmonary histopathology associated with disorders of surfactant homeostasis. Pathologic findings in eonates with mutations in SFTPB (A), SFTPC (B), and ABCA3 (C) are consistent with varying pathologic diagnoses, for example, childhood interstitial pneumonitis (CIP), nonspecific interstitial pneumonitis (NSIP), desquamating interstitial pneumonitis (DIP) or pulmonary alveolar proteinosis (PAP) (D). Severe alveolar remodeling, alveolar loss, macrophage infiltration, varying degrees of alveolar proteinosis, and stromal thickening are observed. In contrast, auto-antibodies against GM-CSF or mutations in the GM-CSF receptor (CSFR2A) are associated with pulmonary alveolar proteinosis in which surfactant lipids and proteins accumulate in the alveolus. Alveolar structure is generally well maintained in PAP (Reproduced with permission from Whitsett JA, Wert SE, Trapnell BC: Genetic disorders influencing lung formation and function at birth. Hum. Mol. Genet. 2004;13:R207–R215.)
Surfactant Protein C (SP-C)
In humans, SP-C is encoded by a single gene (SFTPC, OMIM 178620), located on human chromosome 8.2,10 SP-C mRNA is expressed exclusively in type II epithelial cells in the lung and is translated to produce a 22-kDa precursor that is palmitoylated and proteolytically processed during intracellular transport to form the active, hydrophobic peptide of 35 amino acids stored in lamellar bodies.2,3 After secretion, SP-C enhances the surface-active properties of lipid mixtures, lowering surface tension during compression, and enhancing adsorption rate of lipid films at the air–water interface. Both SP-C and SP-B enhance the speed of formation and stability of lipid films. A mixture of surfactant lipids and proteins SP-B and SP-C improves lung inflation and compliance and is useful for treatment of respiratory distress syndrome (RDS) in newborn infants. SP-C is palmitoylated on cysteine residues near the NH2 terminus. The surface activity of depalmitoylated SP-C is somewhat less than that of palmitoylated SP-C, likely related to reduced stability of the α-helical domain that anchors the peptide within the lipid bilayer. Although the orientation of the palmitoyl groups in a lipid environment is not currently known with certainty, the lipid moiety on SP-C enhances the hydrophobicity of the amino-terminal region enabling its close contact with multilayered lipid films and likely serves to stabilize the α-helical, hydrophobic domain of SP-C. SP-C enhances the uptake of lipids by type II alveolar cells and plays an important role in lipid homeostasis in the alveoli.
In a lipid bilayer, the orientation of the α-helical segment of SP-C is closely parallel with the lipid acyl chains, implying a transbilayer orientation. In a surface monolayer, SP-C has a preferential orientation parallel to the interface, as observed by circular dichroism of monolayer films. The positive charges near the NH2 terminus of SP-C may promote binding of phospholipid vesicles to the monolayer, a step required for insertion of phospholipids into the monolayer. SP-C forms well-defined domains within DPPC/DPPG films below the phase transition temperature of the bulk lipid. SP-C alters the size and shape of lipid vesicles, disrupting vesicular structures, causing the formation of larger vesicles and discoid particles.
Surfactant lipid films fold as a consequence of dynamic compression during the respiratory cycle in a process enhanced by SP-C and SP-B, each interacting with lipids in distinct ways. SP-B serves to stabilize the membrane-to-membrane interactions between the folded lipid layers to create multilayers.3
Role of SP-C in the Pathogenesis of Pulmonary Disease
Deletion of Sftpc in transgenic mice perturbed surfactant function and caused severe interstitial lung disease (ILD) with advancing age.17 While Sftpc−/− mice survive after birth, the mice develop emphysema, pulmonary inflammation, and abnormal lipid accumulations in alveolar macrophages, epithelial, vascular, and stromal cells. Surfactant lipid spreading and stability are only modestly perturbed in the absence of SP-C in vivo. The severity of pulmonary disorder related to SP-C deficiency in mice is strongly influenced by genetic strain, age, and other injuries, indicating that both genetic and environmental factors influence lung structure and function in the absence of SP-C. SP-C binds bacterial endotoxin, supporting a role in innate host defense in the lung. SP-C–deficient mice are susceptible to viral and bacterial pathogens and develop severe pulmonary injury in mouse models of pulmonary fibrosis. The finding that Sftpc−/− mice develop an interstitial pulmonary disorder is consistent with findings in humans, wherein SFTPC mutations cause both acute and chronic lung disease.
Mutations in SFTPC Cause Severe Interstitial Lung Disease in Humans
Mutations in SFTPC represent a rare cause of acute and chronic lung disease in humans.18,19 SFTPC mutations are generally inherited as an autosomal dominant gene that has been causally linked to acute respiratory disease in newborn infants and more commonly, to chronic ILD in infants, children, and adults. De novo mutations in the SFTPC gene have been reported. The diagnosis of SFTPC-related lung disease (OMIM 610913) is usually made during infancy, but can present later in life, the severity of disease varying in a single extended family.19 Most mutations occur in the C-terminal BRICHOS domain of proSP-C that serves as an intramolecular chaperone for the metastable membrane-spanning helical domain.20 The mutant proSP-C protein is misfolded and/or misrouted resulting in intracellular accumulation. Most mutations result in the lack of synthesis of the active SP-C peptide that may influence the pathogenesis of lung disease. Various forms of ILD have been associated with the disease, including acute RDS in newborns, CPI, nonspecific interstitial pneumonitis (NSIP), and other forms of idiopathic pulmonary fibrosis (IPF) (Fig. 5-3).16 Lung histopathology associated with SFTPC mutations is likely influenced by age, duration and severity of the disease, treatment, and both genetic and environmental factors. Infants with SFTPC mutations often present with severe respiratory signs and symptoms following viral infections. Definitive diagnosis is made by identification of mutations in the SFTPC gene. The onset and severity of pulmonary disease in humans is highly variable, even in the same kindreds, indicating that genetic and environmental factors strongly influence the disorder. At present, there is no effective therapy for SP-C–related disease. Lung transplantation has been offered for treatment of hereditary SFTPC deficiency in patients with respiratory failure. Mutations in SFTPC are a rare cause of acute and chronic ILD. More than 50 distinct mutations in SFTPC have been associated with clinical lung disease and include missense, frameshift, splice, insertions, and deletions that generally disrupt the structure of the C-terminal BRICHOS domain. The most common mutation, I73T, is found in more than one-third of patients.
ABCA3 Mutations Cause Respiratory Failure at Birth
ABCA3 is a large, membrane-spanning transport protein that is present in the limiting membrane of lamellar bodies in type II epithelial cells (Fig. 5-2). More than 140 different mutations associated with severe lung disease in newborn infants have been identified, ABCA3 mutations (OMIM 610921) representing the most common genetic cause of neonatal respiratory failure.1,4,21,22 While expressed in many tissues, patients with mutations in ABCA3 present with isolated lung disease, and abnormalities in other organ structures or functions have not been observed. Pathologic findings in newborn infants with respiratory failure are similar to those in mice wherein ABCA3 has been genetically deleted. ABCA3-related lung disease is generally inherited as an autosomal recessive disorder (Table 5-1). Affected infants present with severe respiratory failure characteristic of surfactant deficiency within the first days of life. Their lung disease is refractory to conventional therapies, resulting in respiratory failure and death within the first months of life. Pathologic findings are similar to those in SFTPB-related disease, and include alveolar proteinosis, lipoid pneumonia, cuboidal epithelial cell hyperplasia, interstitial thickening, loss of normal alveolar structures, and features of DIP (Fig. 5-3).16 Older children present with features of NSIP. In newborn infants, respiratory failure is not responsive to surfactant replacement. Lung transplantation has been offered to some patients. ABCA3 is a member of the ATP-dependent, Walker domain–containing proteins that comprise a family of membrane-associated transport proteins that includes the cystic fibrosis transmembrane conductance regulator (CFTR). ABCA3 mediates PC and PG transport into lamellar bodies. The diagnosis of ABCA3-related lung disease is confirmed by nucleotide sequencing of the gene in infants and children with refractory pulmonary disease. While most mutations cause respiratory failure in infancy, the E292V mutation is associated with less severe lung disease, these patients often presenting with ILD later in childhood. Ultrastructural analysis of lung tissue from patients with ABCA3 mutations usually demonstrates the presence of small, atypical lamellar bodies in type II epithelial cells and the absence of tubular myelin in the airways, indicating an abnormality in both intracellular and extracellular lipid homeostasis. The processing of proSP-B is disrupted in some patients with ABCA3-related lung disease.13
Role of TITF1 in Surfactant Homeostasis
TITF1, encoding the homeodomain-containing nuclear transcription factor, thyroid transcription factor-1 (TTF-1), plays a critical role in lung morphogenesis and the expression of SPs.23,24 TTF-1 is expressed in the central nervous system, thyroid, and lung and is required for lung formation during embryonic development.24 TTF-1 regulates the SP genes (SFTPA, B, C, and D), ABCA3, SLC34a2, all expressed in alveolar type II epithelial cells.23 SLC34a2, is a phosphate transporter associated with the disease pulmonary alveolar microlithiasis.25 Mutations in TITF1 have been linked to disorders of the central nervous system, thyroid, and lung (OMIM 600635), and more than 150 patients have been reported to date.26,27 TTF-1–related lung disorders are generally inherited as heterozygous mutations resulting in lung dysfunction of varying severity, ranging from disordered alveolar morphogenesis, surfactant deficiency with respiratory failure in neonates and infants, and ILD in older patients. The majority of patients with TITF1 mutations present with severe lung disease, approximately half of which have a spectrum of brain, thyroid, and lung disease. Histologic findings vary greatly with severe abnormalities in the alveoli, and variable loss of SPs and lipids. Pulmonary disease associated with TITF1 mutations is frequently accompanied by congenital hypothyroidism. The severity of TITF1-related CNS, thyroid, and pulmonary disease varies widely in patients with TITF1 mutations. Diagnosis is made by identification of mutations in the TITF1 gene.