Surfactant Homeostasis: Composition and Function of Pulmonary Surfactant Lipids and Proteins

Introduction

In vertebrates, adaptation to a nonaqueous respiratory environment was achieved by the development of lungs, which provide an extensive surface area for gas exchange. The unique physicochemical boundary between respiratory gases and the alveolar epithelium creates a region of high surface tension, generated by the unequal distribution of molecular forces on water molecules at the air-liquid interface. Unopposed, this surface tension creates collapsing forces that cause atelectasis and respiratory failure. Pulmonary surfactant creates lipid layers separating alveolar gas from the aqueous phase, decreasing these surface forces. It is not surprising that pulmonary surfactant is found in all air-breathing vertebrates studied, including animals as phylogenetically divergent as the lungfish and humans.

Synthesis and secretion of an abundance of phospholipid-rich material accompany the maturation of the lung before birth. The lack of pulmonary surfactant in premature infants results in respiratory distress syndrome (RDS) after birth. Likewise, loss of surfactant function related to lung injury causes acute respiratory failure postnatally. Hereditary disorders of surfactant homeostasis cause respiratory failure in newborn infants and children. The specifics of structure and function of the surfactant complex have important implications for diagnosis and treatment of RDS and other pulmonary diseases. This chapter considers the maturation and function of the pulmonary surfactant system that is required for adaptation to air breathing after birth.

Forming the Gas-Exchange Region of the Lung Parenchyma

The vertebrate lung is derived from epithelial progenitor cells from the anterior foregut endoderm that proliferate and branch within the splanchnic mesenchyme early in gestation. Complex paracrine signaling among diverse pulmonary cells directs stereotypic branching of conducting airways that end in acinar tubules that dilate in late gestation, forming the peripheral saccules that will create the alveolar gas-exchange region after birth. Prior to birth, the stromal-mesenchymal components of the lung thin, and pulmonary capillaries expand as the pulmonary circulatory system comes in close apposition to the epithelial cells lining the peripheral saccules. Two distinct, differentiated epithelial cells, alveolar type 2 cells (AT2), and type 1 cells (AT1) line the peripheral lung saccules. AT1 cells are highly squamous that, together with endothelial cells, create the efficient gas-exchange structure in the alveoli; cuboidal AT2 cells cover much less of the alveolar surface but are critical for the synthesis and secretion of surfactant lipids and proteins needed to reduce surface tension, thereby enabling ventilation ( Fig. 75.1 ). The signaling and transcriptional processes directing branching morphogenesis and lung maturation near the time of birth are increasingly understood. The structural and biochemical maturation of the lung prior to birth include the interactions of multiple cell types, including diverse fibroblasts, myofibroblasts, pericytes, and endothelial cells that interact with epithelial progenitors in a precisely orchestrated temporal-spatial pattern to create the gas-exchange region critical for survival after birth. Recent single cell RNA studies identify more than two dozen major cell types comprising the peripheral lung at the time of birth. ^, In humans, the perinatal lung is considered to be primarily in the late “saccular” stage of development. Further septation and elongation of the saccules during the “alveolar” stage occur after birth, resulting in the formation of the mature alveoli. Many of the nuclear transcription factors and signaling processes involved in early branching morphogenesis also play critical roles in the differentiation of the respiratory epithelium prior to birth. For example, FOXA1, FOXA2, FOXP1/P2, NKX2-1, SOX9, Wnt/β-catenin, GATA6, and CEBPα all play critical roles in the growth and differentiation of epithelial cells in peripheral lung, and therefore influence both lung architecture and pulmonary surfactant homeostasis. ^{, ,} Likewise, glucocorticoid signaling in mesenchymal cells of the lung plays an important role in the maturation of the respiratory epithelium, a process underlying the successful clinical use of antenatal maternal glucocorticoid therapy to enhance fetal lung maturation prior to preterm birth. ^, Since the processes of lung maturation are regulated in precisely timed sequences and occur relatively late in gestation, preterm infants are often born at a time in which neither lung structure nor surfactant homeostasis is adequately developed to support normal ventilation after premature birth.

Surfactant Deficiency and Respiratory Distress Syndrome

Seminal studies by Avery and Mead defined the critical role of pulmonary surfactant in the pathogenesis of RDS in preterm infants that led to the elucidation of the biochemical and physiologic requirements for the synthesis and function of pulmonary surfactant. Lungs from preterm infants dying from RDS lacked the lipid rich material needed to reduce surface tension at an air-liquid interface. Pulmonary surfactant is composed primarily of lipids present in distinct macromolecular aggregates whose structural forms are conferred by the relative abundance of surfactant associated proteins and phospholipids, as well as by the impact of mechanical forces on the surfactant material accompanying the compression and decompression during the respiratory cycle. Tubular myelin, the most abundant structural form of surfactant, is a highly surface-active material that sediments at relatively low gravitational forces and consists primarily of phospholipids and proteins; it is lacking in infants with RDS ( Fig. 75.2 ). Tubular myelin serves as a reservoir from which multilayered lipid films that spread over the alveolar surface are formed. Differentiation of type II epithelial cells and associated production of both surfactant lipids and proteins are incomplete in many preterm infants.

Synthesis, Secretion, and Catabolism of Surfactant

Surfactant lipids are synthesized, stored, secreted, and recycled by type II epithelial cells ( Fig. 75.3 ) (see also detailed reviews by Agassandian and Mallampalli, Goss and colleagues, and Whitsett and colleagues ). Metabolic substrates for lipid synthesis are derived from precursors taken up from the circulation, by de novo synthesis, by reuptake of lipids by type II epithelial cells, and from products of lipid degradation by alveolar macrophages. Within type II epithelial cells, lipids are synthesized in the endoplasmic reticulum (ER) and transferred to Golgi bodies. Alternatively, transport may be mediated by lipid transfer proteins or by direct contact of lamellar bodies (LBs) with the ER, for review see work by Brandsma and Postle. Phosphatidylcholine (PC) transfer to LBs requires an adenosine triphosphate (ATP)-binding cassette transporter A3 (ABCA3), located on the limiting membrane of the LBs. Surfactant proteins (SP) proSP-B and proSP-C are synthesized and transported via the ER and proteolytically processed during transport to LBs. The small, hydrophobic active peptides, mature SP-B and SP-C, are assembled with surfactant phospholipids into membranes that are stored in LBs. In contrast, SP-A and SP-D are secreted independently and are assembled into surfactant lipids after secretion. LBs are secreted into the airway via a process stimulated by catecholamines, purinoreceptor agonists, and stretch. Secretory processes are inhibited by GPR116, an orphan G protein–coupled receptor located on respiratory epithelial cells. ^, After secretion, LBs unwind and interact with SP-A and SP-D to produce tubular myelin and multilayered surface films that spread over the alveolus to reduce surface tension (see Fig. 75.3 ). SP-A and SP-B are required for formation of tubular myelin. ^, The pulmonary collectins, SP-A and SP-D, have important roles in innate host defense in the lung. ^, SP-D regulates extracellular forms of surfactant and has an important role in controlling the size of the surfactant lipid pool. ^, Pulmonary surfactant is recycled, catabolized, or reutilized actively by alveolar type II epithelial cells in a process influenced by SP. Alveolar macrophages play a critical part in surfactant uptake and degradation in a process that depends upon signaling by granulocyte-macrophage colony–stimulating factor (GM-CSF) and its receptors (CF2RA and CF2RB) in alveolar macrophages. ^, Fig. 75.3 provides an integrated schematic of important aspects of the processes critical for surfactant homeostasis in the alveolus.

Metabolic Pathways Regulating Surfactant Production and Homeostasis

Major pathways controlling surfactant lipid synthesis are relatively well established, as recently reviewed in Brandsma and Postle. PC is the most abundant lipid component of pulmonary surfactant, representing approximately 70% of the total lipid content. As the lung matures, the content of PC increases with increasing enrichment of PC 16.0/16.1 (palmitic acid at the C-1 position and palmitoleic acid at the C-2 position). Measurement of disaturated phosphatidylcholine (DSPC) (disaturated PC) by osmium tetroxide has been used as a surrogate for desaturated palmitoyl-phosphatidylcholine (DPPC), the most abundant species in surfactant. DPPC is synthesized de novo by the cytodine diphosphate choline (CDP) choline pathway and by reacylation of PC species in the Land cycle. Fatty acid chains are generated by the enzyme fatty acid synthase, controlled transcriptionally by sterol regulatory element binding proteins (SREBP), that control lipid substrate supply in AT2 cells. The CDP-choline pathway is controlled by choline kinase (CK), phosphocholine cytidylyltransferase (CTP): choline phosphatidyl transferase (CCT), and choline phosphotransferase (CPT), and is activated by peroxisome proliferator–activated receptor (PPAR) γ, stretch, vasoactive intestinal peptide (VIP), and fibroblast growth factor 7. The ABCA3 transporter moves lipids into the lamellar body from the ER, selectively enriching for DPPC. Steady state cellular PC content is further maintained by the basolateral secretion of lipid into the systemic circulation and lymphatics in a process mediated by the transport protein ABCA1, which removes excess cholesterol ester and PC, further enriching unsaturated species to enhance DPPC content in the AT2 cell and surfactant. Enrichment of PC with palmitate is maintained by acyl remodeling via phospholipase A2, peroxiredoxins 6 (Prdx6), and selective removal of unsaturated species by ABCA1 and LPCAT (lysophosphatidylcholine acyltransferase), which selectively incorporates palmitoyl CoA into DSPC.

Biophysics of Pulmonary Surfactant

The close apposition of alveolar type I epithelial cells to pulmonary endothelial cells lining the capillaries forms a highly diffusible air–blood barrier across which gas exchange occurs. The stabilization of alveolar structure during breathing-induced expansion and contraction is achieved by the formation and maintenance of a phospholipid-rich film that spreads over the thin liquid layer (the aqueous hypophase) that covers the alveolar epithelial cell surface (recently reviewed by Autilio and Perez-Gil ). The unique biophysical properties of surfactant prevent alveolar collapse (atelectasis) at low lung volumes by reducing surface tension, which is generated by the aqueous hypophase, to very low levels (<2 mN/m). During alveolar expansion, surface tension increases (to a maximum of 20 to 25 mN/m), stabilizing the alveolus at higher lung volumes. The unique biophysical properties of the lipid films are directly related to the incorporation of DPPC, a saturated phospholipid that allows acyl chains to be very tightly packed as the film is compressed during exhalation. Incorporation of small amounts of cholesterol and other phospholipids with the unsaturated acyl chains helps to maintain the fluidity of the surface film at body temperature. Surfactant proteins SP-B and SP-C facilitate remodeling of the structure of newly secreted surfactant membranes by promoting the incorporation and spreading of lipids as the surface film expands during inhalation. Neonatal lethality in knockout mice and the severe lung disease in patients with mutations in the SFTPB gene indicates that SP-B is indispensable for this process. ^, While SP-C deficiency is not lethal, in mice, SP-C enhances lipid spreading and is required for optimal function of the surface film. Lipid–protein complexes are removed from the surface film during compression and are degraded by alveolar macrophages or are recycled in type II epithelial cells; the recycling process depends at least partly on SP-D, which enhances uptake of surfactant lipids by type II epithelial cells. Maintenance of the surface film is a highly dynamic process that requires integration of synthesis and assembly, secretion, recycling, and degradation. Dysregulation can lead to alterations in the size, composition, or both of the alveolar surfactant pool, resulting in pulmonary alveolar proteinosis (PAP—surfactant accumulation) or (RDS—surfactant insufficiency). Thus, sensing the size and composition of the alveolar surfactant is essential for alveolar homeostasis.

Composition of Surfactant

The composition of pulmonary surfactant is, in general, well conserved among diverse species. The general composition of mammalian surfactant ^, is represented in Fig. 75.4 . Surfactant is composed primarily of phospholipid (predominantly PCs), which represents approximately 80% to 90% of its mass; proteins generally contribute less than 10% of its mass. Lesser amounts of glycolipids and neutral lipids are detected in approximately equal amounts. Phospholipid is the primary surface tension–lowering component of pulmonary surfactant. The phospholipids form multilayered sheets that are derived from tubular myelin or other aggregate forms present in the alveolus. PC is uniquely enriched in disaturated forms of dipalmitoylphosphatidylcholine ( Fig. 75.5 ). In human surfactant isolated from lung minces, PC represents 80% of the total phospholipid, of which 70% is present as the palmitoylphosphatidylcholine; 55% of this lipid species is in the form of disaturated palmitic acid acyl groups or DSPC. Phosphatidylglycerol is also uniquely enriched in pulmonary surfactant, generally representing 5% to 10% of surfactant phospholipids. Phosphatidylglycerol also is capable of reducing surface tension at an air-liquid interface; however, its precise role in surfactant function remains unclear. Other phospholipids, including phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin, are present in relatively low amounts in pulmonary surfactant. Glycolipids also are present in pulmonary surfactant and have been partially characterized in rabbit surfactant. Neutral lipids are present primarily as cholesterol esters and acylglycerol fatty acids. The biologic functions of these components, present in relatively low amounts, have not been determined with certainty. ^{, , ,}

The molecular structures of PC and phosphatidylglycerol are represented in Fig. 75.5 . Several aspects of their structures are critical for surface tension reduction at the alveolar-air interface. Each molecule consists of a three-carbon glycerol backbone. The C ₁ carbon is modified by the addition of polar head groups (relatively more hydrophilic residues). In the case of pulmonary surfactant, the most abundant head groups are choline and glycerol. The C ₂ and C ₃ carbons of the glycerol backbone contain acyl groups of long-chain fatty acids, which are highly hydrophobic and lacking in significant charge. The polar head groups—choline, glycerol, and inositol—of the phospholipids produce charge-dependent interactions among neighboring phospholipid molecules and with water. By contrast, the acyl groups are energetically more stable in a nonaqueous environment and are tightly associated with neighboring phospholipid molecules by interactions between carbon and hydrogen atoms of the acyl chains. Hence these molecules are inherently insoluble in aqueous environments and form a variety of complex structures that include membrane monolayers, bilayers, multilayers, micelles, inverted micelles, and vesicles.

The surface properties of surfactant phospholipids (spreading, stability, and surface tension reduction) are influenced by a number of factors, including the SP SP-B and SP-C, and the degree of saturation of the acyl chains, which alter the tightness of packing of phospholipid molecules in membranes. The surface activity of surfactant is readily inhibited by serum proteins, blood, edema fluid, or non-SP derived from lung injury, so maintenance of alveolar-capillary stability is critical for maintaining surfactant function. The fatty acid composition of the phospholipids in pulmonary surfactants has been determined for various species. The structure of the acyl chains and the composition of the major phospholipids are important determinants of the organization of the membranes. PC isolated from pulmonary surfactant is uniquely enriched in forms with disaturated palmitic acid (C16) acyl chains. Enrichment of these phospholipid species at the surface results in densely packed lipid sheets, creating an interface with extremely low surface tension. Saturated acyl chains contain no methylene (C = C) bonds, and the carbon atoms are fully hydrogenated. Membranes containing such lipids pack densely through the hydrophobic interactions of the acyl chains. The ordering of phospholipid molecules in the surfactant membrane also is highly dependent on temperature. Surfactant lipids are present in a gel or crystalline state at the physiologic temperatures of homeothermic organisms because the transition temperature (temperature of melt) of DPPC is approximately 41°C. Therefore, DPPC would be present in a relatively rigid state at 37°C. However, the presence of minor lipids, proteins, and unique phospholipid acyl chains alters the packing characteristics of the phospholipids. The relative abundance of the major lipid classes and their acyl chain length and specific composition, including the proportion of molecular species with unsaturated acyl chains, therefore result in a unique pulmonary surfactant mixture that may alter the surface properties of the surfactant film. The characteristics of rapid adsorption and stability during compression of pulmonary surfactant are not properties inherent in the phospholipids alone, but require the presence of the SP SP-B and SP-C. The hydrophobic SP SP-B and SP-C are required for full surface-active properties of the lipids in surfactant and are active components of surfactant replacement preparations used to prevent or treat RDS in preterm infants.

Composition of Lamellar Bodies

Surface-active material can also be isolated from its primary intracellular storage site in LBs of alveolar type II cells. LBs are lysosomal-like organelles highly enriched in phospholipids, generally containing approximately 10 to 12 mg of phospholipid per mg of protein. A diversity of proteins are present in the LBs as identified by mass spectroscopy. The limiting membrane of the lamellar body contains at least one ABC transporter, ABCA3, which plays an important role in importing phospholipids into the lamellar body. Mutations in the ABCA3 transporter block the formation of LBs and cause severe lung disease in newborn infants. ^, Lipid composition of LBs is similar to that for surfactant isolated from lung lavage fluid. The active, fully processed SP-B and SP-C peptides are highly enriched in LBs and are co-secreted with phospholipids into the air space. Like ABCA3, mutations in the genes encoding SP-B and SP-C ( SFTPB and SFTPC ) cause severe lung disease in neonates and infants.

Developmental Changes in Phospholipid Composition

The phospholipid composition of alveolar lavage material changes during perinatal development. Increased phospholipid synthesis and secretion occur with advancing gestation and are influenced by a variety of hormonal and cellular factors. Because pulmonary secretions contribute a significant volume to amniotic fluid, increased phospholipid in amniotic fluid accompanying advancing gestation has been used for determining the relative maturity of the fetal lung and thus predicting the risk of RDS in premature infants. The PC content of amniotic fluid increases during the last third of human gestation. The ratio of lecithin (PC) to sphingomyelin, otherwise known as the L/S ratio , has been useful in the clinical assessment of risk for RDS. Surfactant content in amniotic fluid can be determined by a number of procedures that predict pulmonary maturity. Various amniotic fluid assays are useful in predicting surfactant function or lack of respiratory distress in preterm infants, including the L/S ratio; lamellar body counts; quantitation of phosphatidylglycerol, DSPC, or PC; and fluorescence anisotropy. Changes in total phospholipid content and in the relative abundance of phospholipid species also accompany respiratory failure in infants and adults.