Surfactant Protein-A

SP-A monomer is a water-soluble 24-kDa protein that is heavily glycosylated to yield a protein of about 36 kDa. SP-A monomers contain a collagenous domain that assembles into a collagen-like triple helix to form a trimer; six trimeric subunits associate to form a multimeric protein with a molecular mass of about 650 kDa (12)(Figure 8-2). SP-A associates primarily with saturated phosphatidylcholine and is associated with tubular myelin, a unique square lattice membrane structure that may be involved in host defense. Surfactant from mice that completely lack SP-A does not form tubular myelin, but the mice have normal lung function (13). The absence of SP-A in mice also does not disrupt the secretion, clearance, or catabolism of surfactant lipids.

Figure 8-2.

Surfactant Protein Structures SP-A and SP-D are collectins that each contain a collagen-like domain (CLD) and a carbohydrate recognition domain (CRD). The CLD facilitates formation of an SP-A octadecamer and an SP-D dodecamer. The dashed line represents the signal peptide, the black line is the NH₂-terminal domain, and the yellow line is the neck region. SP-B and SP-C are synthesized as proproteins that are proteolytically processed to very hydrophobic mature peptides that associate with surfactant membranes: Mature SP-B forms amphipathic helices that associate with the surface of the membrane, whereas mature SP-C forms a metastable α helix that spans the membrane bilayer. Black represents the signal peptide, green is the NH₂-terminal propeptide domain, red is the mature peptide, and blue is the COOH-terminal peptide domain.

The major function of SP-A is as an innate host defense protein and regulator of inflammation in the lung (14). SP-A binds to gram-positive and gram-negative bacteria, viruses, and fungi primarily through its carbohydrate recognition domain (CRD). SP-A facilitates phagocytosis of pathogens by macrophages and may directly kill some microbes via membrane permeabilization. SP-A can inhibit inflammation induced by lipopolysaccharide (LPS), peptidoglycan, or zymosan by binding to cell surface receptors (e.g. CD14 and TLR2) for these pathogen-derived molecules. Binding of SP-A to SIRPα also inhibits pro-inflammatory signaling. SP-A can also evoke a context-dependent pro-inflammatory response by binding to rough LPS (via the CRD) and CD14 (via the neck region) or to calreticulin/CD91 on the surface of macrophages via the collagen-like domain. SP-A levels are low in surfactant from preterm lungs and increase as the type II cell numbers increase and mature. The fetal lung increases expression of SP-A in response to corticosteroid or chorioamnionitis in animal models (15).

SP-D

SP-D is a collectin that shares structure and innate host defense functions with SP-A (12). The 43 kDa monomer also forms trimers through its collagen-like domain. Twelve monomers associate to form a multimeric protein composed of four trimeric subunits. SP-D expression in the lung is restricted primarily to type II epithelial and Clara cells, but it is widely expressed by other epithelial cells in the body. SP-D is present in lower amounts in bronchoalveolar lavage than SP-A and is primarily recovered in the water-soluble fraction. As for SP-A, SP-D binds to pathogen-associated molecular patterns (PAMPs) through its CRD; facilitates uptake of bacteria, viruses, and fungal pathogens by macrophages; and may directly kill some pathogens by membrane permeabilization (11). Although SP-D does not appear to interact with intracellular surfactant synthesis, storage and secretion pathways, it does bind to phosphatidylinositol. Absence of SP-D in mouse models is associated with altered surfactant metabolism and a greatly increased alveolar surfactant pool size. SP-D deficient mice also have progressive emphysema related to increased oxidant stress associated with altered macrophage function (16). Addition of SP-D to surfactant used to treat preterm sheep decreases ventilator-mediated inflammation (17). SP-D expression is increased by antenatal corticosteroids and by fetal exposure to inflammation in animal models. Exposure to both corticosteroids and inflammation further increases expression (15).

SP-B

SP-B is a hydrophobic peptide of 79 amino acids that is cleaved from a precursor protein of approximately 40 kDa prior to association with surfactant lipids in type II epithelial cells (18). Surfactant lipids and proteins (SP-B and SP-C) are stored as concentric bilayer membranes (lamellae) in specialized secretory organelles (lamellar bodies) prior to secretion into the alveolar airspaces. The ability of SP-B to both lyse and fuse bilayer membranes is critical for organization of surfactant membranes within lamellar bodies and for transition of newly secreted membranes to a surface active film at the alveolar air–liquid interface. SP-B is absolutely essential for lung function as knockout mice have normal lung structure at birth, but cannot initiate air breathing because of a lack of functional surfactant (19). In the absence of SP-B, type II cells fail to form lamellar bodies or process SP-C. Thus, SP-B is required for both the synthesis and assembly of surfactant in type II cells as well as for its function in the alveolar compartment. SP-B comprises only about 2% of surfactant, and when this amount is decreased in adult mice, the animals develop lung injury and respiratory failure (20). Production of large amounts of appropriately folded active synthetic SP-B peptide has not been achieved, but shorter peptide mimetics have been developed for use in synthetic surfactant preparations with some success. As with the other surfactant components, SP-B expression and amounts increase with advancing gestational age and increase with antenatal corticosteroid or fetal exposure to inflammation (15).

SP-C

SP-C is the other hydrophobic protein in surfactant that comprises up to 4% of surfactant. Organic solvent extracts of surfactant recover both SP-B and SP-C with lipids. Like SP-B, SP-C is synthesized as a precursor protein that is processed to an extremely hydrophobic 35 amino acid (4 kDa) protein that associates with lipids in lamellar bodies (18). The mRNA for SP-C appears in the epithelium of the developing airways from early gestation. With advancing lung maturation, SP-C expression is restricted to type II epithelial cells. The amino acid sequence and cellular localization of SP-C are remarkably similar across mammalian species consistent with an important, but poorly understood, function. Although mice that lack SP-C have normal lung structure and surfactant function at birth (21), these animals develop strain-dependent, progressive interstitial lung disease (ILD) and emphysema as they age. Although not critical for survival, SP-C contributes to surface film stability by interacting with SP-B and lipids, thus contributing to the unique surface properties of surfactant. SP-C expression increases in the fetal lung as type II cell numbers and maturation increase.

Synthesis and Secretion

Type II cells and macrophages are the cell types responsible for the major pathways involved in surfactant metabolism (Figure 8-3). The synthesis and secretion of surfactant is a complex sequence of biochemical synthetic events that results in the release by exocytosis of lamellar bodies to the alveolus (18). The basic pathways for the synthesis of phospholipids are common to all mammalian cells. Specific enzymes use glucose, phosphate, and fatty acids as substrates for phospholipid synthesis. Surfactant lipids are synthesized primarily in the endoplasmic reticulum (ER) and transported to lamellar bodies, the storage organelle for surfactant. An important component of the pathway involved in the intraorganelle transfer of newly synthesized surfactant lipids is ABCA3, a phospholipid import protein located in the limiting membrane of the lamellar body. The orientation of ABCA3 in the membrane and the absence of typical lamellar bodies in ABCA3 null mice strongly suggest that surfactant phospholipids are transported directly from the ER to the lamellar body, via as yet unidentified lipid transfer proteins located in the cytosol of type II cells. Candidate phosphatidylcholine transfer proteins and other potential lipid importers have been identified in the lamellar body proteome, but the involvement of these proteins in surfactant biosynthesis has yet to be evaluated (22). In the adult lung, surfactant secretion is presumed to be regulated by the microenvironment of the alveolus. Secretion can be stimulated by beta-agonists, purinergic agonists, or hyperventilation. In the fetus, surfactant is released into fetal lung fluid in increasing amounts as gestation advances. Surfactant is secreted into fetal lung fluid and is carried out of the lung with fetal breathing and subsequently swallowed or diluted in amniotic fluid. Thus, amniotic fluid can be used to evaluate the development of the surfactant system.

Figure 8-3.

Surfactant metabolism biosynthesis of surfactant likely involves distinct pathways for surfactant proteins and lipids (green arrows). Newly synthesized SP-B and SP-C are trafficked from the ER to lamellar bodies (LB) via the Golgi and multivesicular body (MVB), whereas SP-A and SP-D are constitutively secreted by the type II epithelial cells (dashed green arrows). In contrast, surfactant phospholipids are likely directly transported from the ER to specified lipid importers (ABCA3) in the lamellar body-limiting membrane (solid green arrow). Surfactant proteins and lipids are assembled into bilayer membranes that are secreted into the alveolar airspace, where they form a surface film at the air-liquid interface. Cyclical expansion and compression of the bioactive film results in incorporation (green arrow) and loss (red arrows) of lipids/proteins from the multilayered surface film. Surfactant components removed from the film are degraded in alveolar macrophages or are taken up by the type II epithelial cell for recycling (dashed red arrow) or degradation in the lysosome (solid red arrow). The MVB plays a key role in the integration of surfactant synthesis, recycling, and degradation pathways. Green arrows indicate biosynthetic pathways; red arrows indicate degradation and recycling pathways.

Alveolar Cycle of Surfactant

After exocytosis of lamellar bodies by type II cells, surfactant proceeds through a series of form transitions that define its metabolic and functional roles. The exocytosed lamellar bodies “unravel” into the elegant “tubular myelin” structure with SP-A at the corners of the lattice (Figure 8-3). Tubular myelin requires at least SP-A, SP-B, and the phospholipids for its unique structure. Tubular myelin and other loose, less-structured surfactant lipoprotein membrane arrays are the pool in the fluid hypophase that supplies surfactant for the surface film within the alveolus and small airways. Compression of this film squeezes out unsaturated lipids and some protein components of surfactant with concentration of saturated phosphatidylcholine in the surface film. New surfactant enters the surface film from tubular myelin and the loose lipid arrays, and “used” surfactant leaves as small vesicles, which then are cleared from the airspaces. The major differences in composition between the surface-active surfactant structures and the small vesicular forms are that the small forms contain SP-D but little SP-A, SP-B, or SP-C (23). The small vesicles of used surfactant are much less surface active and seem to be the major catabolic form of the lipids that are taken up by type II cells and by alveolar macrophages for recycling or catabolism.

Alveolar macrophages are the sentinel immune cells of the lung. These cells are in the airspaces directly in contact with the alveolar hypophase and surfactant. Fetal monocytes seed the developing lung and undergo granulocyte-macrophage colony-stimulating factor (GM-CSF) mediated differentiation to alveolar macrophages shortly after birth (24). Once differentiated, alveolar macrophages have a relatively long life span under normal conditions. Important functions of alveolar macrophages include immune surveillance, phagocytosis of invading microorganisms, antigen presentation, interactions with adaptive immune cells, and surfactant homeostasis. Fetuses have very few alveolar macrophages. In mice, primitive macrophages can be detected in the lung interstitium from early gestation, while in other species including nonhuman primates and sheep, few macrophages are found in the fetal lung (25). Fetal exposure to inflammation and lung injury can mature lung monocytes into macrophages and stimulate their migration into the fetal alveolar spaces (26).

The number and maturity of type II epithelial cells increase in the fetal human after about 20 weeks gestation. Immature type II cells with large intracellular lakes of glycogen mature with the disappearance of glycogen and the appearance of more and larger lamellar bodies. Just preceding and following birth, lamellar bodies are secreted to yield an alveolar pool that is primarily lamellar bodies and tubular myelin (27). This surfactant then begins to function with aeration of the lung. As the newborn goes through neonatal transition, the percentage of surface-active forms falls, and the small vesicular forms increase. At equilibrium approximately 50% of the surfactant in the airspaces is in a surface-active form, and 50% is in the inactive vesicular form. Conversion from surface active to inactive surfactant forms occurs more rapidly in the preterm, presumably because there is a lower pool size and less-effective surfactant with lower amounts of the surfactant proteins. Pulmonary edema can further accelerate the process, with the net result being a depletion of the surface-active fraction of surfactant despite normal surfactant pool sizes.

Surfactant Turnover

The Adult Lung

In the adult human, the amount of surfactant in the airspaces recoverable by bronchoalveolar lavage is about 5 mg/kg body weight (28). There are no reliable measurements of turnover of the surfactant components in the normal adult human. In animal models (primarily the rabbit) the airspace pool size of surfactant is about 16.5 mg/kg, and the lamellar body pool size is about 24 mg/kg (Figure 8-4). The kinetics of secretion were measured with radiolabeled precursors of surfactant components. Clearance kinetics were measured using radiolabeled surfactant components given into the airspaces (29–31). The lag from synthesis to peak accumulation of surfactant lipids in the airspace is about 15 hours. Once secreted, the half-life of the subsequent linear loss from airspace is about 10 hours. The curves have been modeled to demonstrate that the surfactant lipids SP-B and SP-C from the airspace appear in lamellar bodies for resecretion with an efficiency of about 25% (31). The residual surfactant lost from the airspaces is catabolized by alveolar macrophages. Some surfactant moves from the alveoli into small airways, but large amounts of surfactant are not lost up the airways. This elaborate steady-state control of surfactant pool sizes and therefore function in the adult lung can be disrupted by injury to type II cells and by a block in surfactant catabolism by alveolar macrophages from a lack of GM-CSF signaling resulting in alveolar proteinosis (32).

Figure 8-4.

Estimates of pool sizes and flux rates of surfactant in adult rabbits (blue) and in 3-day-old newborn rabbits (red). All values were measured as saturated phosphatidylcholine and have been converted to mg/kg total surfactant (1 μ mol saturated phosphatidylcholine is equivalent to about 1.5 mg total surfactant). The 3-day rabbits weighed about 65 g. The values are from modeling studies using radiolabeled precursors of saturated phosphatidylcholine by Jacobs and colleagues(29–31).

Surfactant Inactivation

Surfactant function is easily disrupted by multiple factors that frequently occur in the preterm lung (Table 8-2). Once the preterm lung has released surfactant stores at delivery, the surfactant will be depleted with time by the normal conversion of the pool from the tubular myelin and loose surfactant arrays into the catabolic liposomes unless there is active replacement. The generation of new secretion capacity from lamellar bodies from de novo synthesis and recycling is a slow process, and efficient recycling depends on normal lamellar body and alveolar pool sizes. The preterm with just enough surfactant at birth to transition to air breathing may become functionally deficient over hours. Further, the surfactant of the preterm lung has lower amounts of saturated to total phosphatidylcholine and lower amounts of the surfactant proteins (46). The multiple proteins, lipids, and other factors that can interfere with film formation listed in Table 8-2 will be more inhibitory for surfactant from the immature lung than mature surfactant that contains more saturated phosphatidylcholine, more SP-B and SP-C, and particularly more SP-A. Finally, surfactant lipids can function as a thromboplastin and promote clotting of plasma that leaks into the airspaces. The surfactant deficient preterm lung is easily injured by spontaneous or mechanical ventilation with increased permeability of the epithelium, resulting in proteinaceous pulmonary edema (46). Plasma components will clot to form hyaline membranes that can trap surfactant, removing it from the functional pool. Multiple substances interfere with film formation by competing with surfactant for the air–water interface. These inactivation phenomena are concentration dependent, as high concentrations of normal surfactant can form stable surface films in the presence of plasma or inhibitors, while surface film formation by low surfactant concentrations in the hypophase are easily disrupted (47). Thus, the preterm lung is at substantial disadvantages because the immature surfactant with functional deficits is likely to be present in small amounts. The term lung also can have surfactant inactivation by meconium and the inflammatory products from pneumonia.

Surfactant Treatment

The treatment responses to surfactant illustrate the effects of surfactant on the developing lung. It must be emphasized that the development of surfactant treatment has transformed the care of preterm infants (48). The reasons that surfactant treatments are so effective go beyond simply acutely improving surface tensions and thus the physiology of the preterm lung. The major reason that surfactant treatments cause persistent clinical responses is that the metabolic characteristics of surfactant phospholipids and proteins in the preterm are favorable (35). Alveolar and tissue pool sizes are small, and the rate of accumulation is slow. Treatment acutely increases both the alveolar and tissue pools because the exogenously administered saturated phosphatidylcholine is taken up by type II cells and processed for resecretion. The surfactants used clinically are not equivalent in composition or function to native surfactant in the mature lung. Furthermore, airway instillation does not achieve an ideal distribution of surfactant. However, within hours following surfactant treatment of preterm animals, the surfactant recovered by alveolar wash has improved function. Therefore, the preterm lung, if uninjured, can rapidly transform surfactants used for treatment with poor function to a better surfactant (47). Also of benefit is the slow catabolic rate of surfactant, with the result being that the surfactant used for treatment remains in the lungs, is recycled, and is not rapidly degraded. The surfactant used for treatment becomes substrate for the endogenous recycling pathways to increase overall surfactant quantities. Treatment doses of surfactant do not feedback-inhibit the endogenous synthesis of saturated phosphatidylcholine or the surfactant proteins (49). No adverse metabolic consequences of surfactant treatment on the endogenous metabolism of surfactant or other lung functions have been identified.

The static mechanics of the preterm lung are strikingly improved by surfactant treatments (Figure 8-5A). The dynamic lung mechanics also are altered by surfactant treatments (35). The time constant for deflation increases, resulting in less-rapid lung emptying. The clinical correlate is that a surfactant treatment can increase the functional residual capacity of infants with RDS by two mechanisms: the improved deflation stability and the longer expiratory time constant. The consistent initial response of infants with RDS to surfactant treatments is a rapid improvement in oxygenation, whereas improvements in PCO₂, compliance, and therefore ventilatory support variables tend to change more gradually. The improved oxygenation without changes in ventilation results from the acute increase in lung volumes following surfactant treatments. In experimental animals, these acute physiological responses are accompanied by much more uniform aeration of the preterm lung at the anatomic level, a decreased lung permeability, and less indicators of lung injury with mechanical ventilation (50,51).

RDS and Induced Lung Maturation

At term the total amount of surfactant present in the human airways plus lamellar body pools probably exceeds that in the adult by about 10-fold on a body weight basis. Lamellar bodies first appear within type II cells by 20 to 24 weeks’ gestation in the human fetus, and the amount of saturated phosphatidylcholine in lung tissue progressively increases to term. Lung maturity as defined clinically by the absence of RDS in the human fetus is generally present after 36 weeks of normal gestation, but infants born at 24 weeks can have “mature lungs” based on their ability to exchange oxygen and CO₂. Therefore, a 12-week window of “early maturation” is possible for the human, in part because the surfactant synthetic and storage machinery can be induced in the human early in gestation.

The clinical syndrome linked to inadequate amounts of surfactant is RDS, but the identification of surfactant deficiency is problematic clinically. There are no tests to measure surfactant amount in the airspaces, and some preterm infants will have sufficient surfactant, but that surfactant may be inhibited by edema or inflammation associated with pneumonia, for example.

From the epidemiologic perspective, the incidence of RDS increases as gestational age decreases. However, incidence of RDS is not easily defined because of variable definitions and the effect of different clinical care strategies on the diagnosis of RDS. For example, from 1997 to 2002 the NICHD Neonatal Research Network defined RDS as the need for oxygen and some ventilatory support, plus a compatible chest roentgenogram. The Network reported the incidence of RDS for infants less than 1 kg as 63% (52). The definition was changed for 2003 to 2007 to the use of supplemental oxygen for >6 hr and 95% of similar infants than had a diagnosis of RDS (53). In contrast, only about 50% of infants with birth gestations less than 28 weeks that are initially supported with CPAP have sufficient RDS to receive surfactant (54). If the use of surfactant is a surrogate for significant surfactant deficiency, then many very early gestation infants did not have severe RDS. Biologically, this indicates that induced lung maturation is very frequent. This spontaneous early lung maturation in the human fetus is believed to result from stress-induced maturation events that can be maternal, placental, or fetal in origin. Surprisingly, the fetal stress that must accompany fetal growth restriction or preeclampsia, does not consistently induce early lung maturation.

A changing epidemiology of RDS results in part from the more frequent clinical use of antenatal glucocorticoids and changes in obstetric practice that delay preterm delivery, presumably allowing the lung to mature. Numerous clinical trials have documented that maternal corticosteroid treatments decrease the incidence of RDS by about 50%, and those infants with RDS tend to have less severe disease (55). Chronic infection and fetal exposure to inflammation and histologic chorioamnionitis is frequent in pregnancies with preterm labor between 22 and 30 weeks gestation and also is associated with a decreased incidence of RDS (56). In experimental models, fetal pro-inflammatory exposures induce striking increases in surfactant and improvements in postnatal lung function without increasing fetal cortisol levels (57). Therefore, fetal exposure to inflammation may have the short-term benefit of increasing surfactant and decreasing RDS.

Fetal plasma cortisol increases as the fetus approaches term and is associated with the large increase in surfactant at term. Corticotropin-releasing hormone-deficient mice and mice with ablation of the glucocorticoid receptor have inadequate surfactant to survive following term birth (58,59). Therefore, endogenous cortisol is essential for normal lung maturation. The very early gestation human fetal lung also is responsive to corticosteroids. Explants of human lung at 14 to 20 weeks gestational age differentiate in organ culture in the absence of hormonal stimuli, and corticosteroids and thyroid hormones accelerate maturation (60).

The responses of the fetal lung to corticosteroids are multiple and impact many different systems that will influence the clinical outcome (61). Biochemical markers of maturation include glycogen loss from type II cells, increased fatty acid synthesis, increased beta receptors, and increased choline incorporation into surfactant phosphatidylcholine. In vivo, animals demonstrate improved lung function and survival. Corticosteroid treatment also decreases the tendency of the preterm lung to develop pulmonary edema (50). Although the primary effect of corticosteroids on the fetal lung is generally considered to be induction of surfactant synthesis, effects on enzymes in the synthetic pathways for surfactant have not been consistently demonstrated, and surfactant pool sizes do not increase until more than 4 days after maternal glucocorticoid treatments in sheep (61). Corticosteroids induce lung structural maturation by decreasing the amount of mesenchyme and increasing the surface area for gas exchange as is reflected by increased lung volumes within 12 to 24 hrs in fetal sheep (62). In preterm animal models, corticosteroid treatment changes the dose-response curve for surfactant treatments such that less surfactant is needed to achieve larger clinical responses (63). Because of increased lung volume, corticosteroid-treated fetuses also have improved responses to postnatal surfactant (64). There are additive or synergistic effects between the corticosteroid-exposed lungs and surfactant treatments in animal models.

Antenatal corticosteroid treatment is now the standard of practice for pregnancies at risk of preterm delivery (55). This therapy is effective and safe, although there is not long-term follow-up for infants born before 28 weeks’ gestation. Repetitive courses of antenatal glucocorticoids have been given at 7- to 10-day intervals, a practice based on the suggestion that the fetal benefit was lost after this interval (61). Maternal glucocorticoid treatments at 7-day intervals in sheep cause fetal growth restriction but augment lung maturation. Randomized trials in women at risk for preterm delivery demonstrate modest benefit, but there is some concern about longer-term outcomes, especially for infants exposed to 4 or more antenatal courses of antenatal corticosteroids (65). There is presently insufficient information for a strong recommendation about the use of repeated corticosteroid treatments.

The only other lung maturation strategy that has been extensively evaluated clinically is the combination of corticosteroids and thyrotropin-releasing hormone (TRH). Thyroid axis hormones induce lung maturation and can act synergistically with corticosteroids in vitro. Thyroid hormones do not cross the human placenta efficiently, but the tripeptide TRH crosses to the fetal circulation and increases fetal thyroid hormone levels. Unfortunately, when evaluated in large randomized, controlled trials, TRH demonstrated no benefit, and possible risks were identified (66).