perez10_Surfactant

Jesús Perez-Gil and Timothy E. Weaver 

Physiology 25:132-141, 2010. doi:10.1152/physiol.00006.2010 

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Lamellar Bodies Form Solid Three-dimensional Films at the Respiratory Air-Liquid 

Interface 

A. Ravasio, B. Olmeda, C. Bertocchi, T. Haller and J. Perez-Gil 

J. Biol. Chem., September 3, 2010; 285 (36): 28174-28182. 

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on the following topics: 

Biophysics .. Metabolism 

Medicine .. Pulmonary Surfactants 

Physiology .. Homeostasis 

Physiology .. Pulmonary Alveoli 

Medicine .. Respiration 

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Pulmonary Surfactant Pathophysiology: 

Current Models and Open Questions 

Pulmonary surfactant is an essential lipid-protein complex that stabilizes the 

respiratory units (alveoli) involved in gas exchange. Quantitative or qualitative 

derangements in surfactant are associated with severe respiratory pathologies. 

The integrated regulation of surfactant synthesis, secretion, and metabolism 

is critical for air breathing and, ultimately, survival. The goal of this 

review is to summarize our current understanding and highlight important 

knowledge gaps in surfactant homeostatic mechanisms. 

PHYSIOLOGY 25: 132–141, 2010; doi:10.1152/physiol.00006.2010 

Jesús Perez-Gil 1 and 

Timothy E. Weaver 2 

1 Department Bioquímica, Faculty Biología, 

Universidad Complutense, Madrid, Spain; and 

2 Cincinnati Children’s Research Foundation 

and University of Cincinnati College of 

Medicine, Cincinnati, Ohio 

jpg@bbm1.ucm.es 

The vital process of mammalian breathing is dependent 

on an extensive gas exchange surface provided 

by the alveoli in the lung periphery. Surface 

tension on the epithelial side of the air-blood barrier 

exerts a collapsing pressure that is stabilized by 

spreading of a lipid-rich film (pulmonary surfactant) 

at the alveolar air-liquid interface. Quantitative 

[e.g., respiratory distress syndrome (RDS)] or 

qualitative [e.g., acute RDS (ARDS)] changes in surfactant 

lead to alveolar collapse and the need for 

ventilatory support; likewise, accumulation of surfactant 

in the alveolar airspaces (e.g., pulmonary 

alveolar proteinosis) impedes gas exchange. Thus 

the integrated regulation of surfactant synthesis, 

secretion, and metabolism is essential for air 

breathing and, ultimately, survival. 

Research in the past three decades has provided 

extensive insight into surfactant biology that, in 

turn, has facilitated development of surfactant replacement 

therapies: the latter have profoundly 

impacted the incidence of morbidity and mortality 

in newborn infants. However, despite the many 

remarkable achievements in this field, major questions 

remain unanswered. The goal of this review is 

to summarize our present understanding of surfactant 

homeostatic mechanisms and highlight important 

knowledge gaps in molecular pathways 

involved in surfactant synthesis, secretion, recycling, 

and degradation. This focus excludes discussion 

of the role of surfactant in innate lung 

defense, and the reader is referred to several excellent 

reviews of this topic (46, 57, 60, 109). 

Synthesis and Assembly 

of Surfactant 

The major components of pulmonary surfactant 

include phospholipids (80%), neutral lipids 

(mainly cholesterol, 10%), and the two hydrophobic 

peptides (1–2%) surfactant protein B (SP-B) 

and SP-C. Type II epithelial cells synthesize and 

assemble the lipid and protein components into 

complexes that are stored as tightly packed membranes 

in lamellar bodies until secreted into the 

alveolar airspaces (FIGURE 1). Molecular pathways 

involved in the regulation of expression, intracellular 

trafficking, and processing of the surfactant 

proteins have been extensively characterized; 

much less is known about the intracellular pathways 

regulating surfactant lipid biosynthesis. 

Surfactant Proteins 

Surfactant Protein B 

SP-B is synthesized as a soluble proprotein that is 

processed to a smaller, lipid-associated peptide in 

the distal secretory pathway within the type II cell. 

Processing of proSP-B occurs in the multivesicular 

body (MVB) and lamellar body (LB) compartments 

and involves the action of napsin A, cathepsin H, 

and pepsinogen C (12, 37, 45, 96); at least one and 

perhaps several other as yet unknown enzymes 

participate in proprotein maturation. The fully 

processed, 79-amino acid mature peptide facilitates 

organization of surfactant membranes in the 

lamellar body, likely through its ability to promote 

membrane-membrane contacts, perturbation of 

lipid packing, and membrane fusion (49, 89). SP-B 

deficiency, arising from mutations in the human 

gene (SFTPB) or disruption of the mouse locus 

(Sftpb), results in vesiculated lamellar bodies with 

few or no bilayer membranes and electron dense 

inclusions. Most importantly, severe SP-B deficiency 

(i.e., 75% reduction in SP-B content in the 

airspaces) results in fatal RDS (19, 67, 70, 71). SP-B 

expression is altered in a number of acute and 

chronic lung diseases and may therefore contribute 

to pathogenesis (107). Thus identification of 

transcriptional pathways that modulate SP-B expression 

and proprotein maturation in both 

healthy and diseased lung remains an important 

area of investigation. 

SP-B is also expressed in non-ciliated, bronchiolar 

epithelial cells (Clara cells). The proprotein is 


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not fully processed to the mature peptide in Clara 

cells, and the processing enzymes and fates of resulting 

peptides are largely unknown. Importantly, 

targeted expression of SP-B in type II epithelial 

cells but not Clara cells of Sftpb / mice is sufficient 

to rescue the neonatal lethal phenotype (62). 

Taken together, these data suggest that the function 

of SP-B in Clara cells is unrelated to surfactant 

homeostasis. Given the presence of saposin-like 

domains in the NH 2 - and COOH-terminal regions 

of the proprotein (77), it is possible that saposinlike 

peptides derived from these regions may play a 

role in innate host defense of the airspaces. Consistent 

with this hypothesis, a saposin-like peptide 

derived from the SP-B propetide was recently identified 

in bronchoalveolar lavage fluid and alveolar 

macrophages. The peptide exhibited antimicrobial 

activity only at acidic pH, suggesting that it promotes 

killing of bacteria internalized by macrophages in the 

airspaces (112). Whether the saposin-like domain in 

the COOH-terminal region of the SP-B proprotein is 

also involved in alveolar defense remains to be 

established. 

Surfactant Protein C 

Unlike SP-B, SP-C expression in the lung is restricted 

to alveolar type II epithelial cells. Newly 

synthesized SP-C is an integral membrane protein 

in which the NH 2 terminus resides in the cytosol 

and the COOH terminus resides in the ER lumen 

(56). The cytosolic domain encodes information 

required for intracellular trafficking of SP-C to the 

MVB/LB compartments (20, 21, 54, 58). The COOH 

terminal, lumenal domain may act as an intramolecular 

chaperone that transiently stabilizes the inherently 

unstable transmembrane domain (TMD), 

presumably until palmitoylation of two cytosolic 

cysteine residues assume this role (44, 52, 53). The 

identity of the palmitoyl acyltransferase that acylates 

SP-C is not known (93). 

Like SP-B, SP-C is synthesized as a proprotein 

that is processed to a smaller, hydrophobic peptide 

in the MVB/LB compartments. The fully processed, 

35-amino acid mature peptide consists of a TMD 

and a short segment of the cytosolic domain. Enzymes 

involved in the maturation of proSP-C have 


FIGURE 1. Biosynthesis of pulmonary surfactant 

Synthesis of the protein and phospholipid components of surfactant may proceed via separate pathways (green 

lines). SP-B and SP-C traffic through the Golgi and late endosome/multivesicular body (MVB) to the lamellar body. In 

contrast, surfactant phospholipids may traffic directly from the ER to the lamellar body; phospholipid transfer protein(s) 

(PLTP) and ABC transporters likely play an important role in phospholipid trafficking. (It is also possible that 

direct contact between the ER and lamellar body may facilitate phospholipid transfer; see text.) Transcriptional pathways 

involved in coordinated regulation of surfactant protein and phospholipid synthesis are indicated by black arrows. 

Surfactant components internalized from the airspaces may also contribute to biosynthesis (red lines). The 

micrograph of the multilayered surfactant surface film was modified from Ref. 88. 

PHYSIOLOGY • Volume 25 • June 2010 • www.physiologyonline.org 133

REVIEWS 

not been conclusively identified, although cathepsin 

H has been implicated (54), suggesting that the 

processing machinery may be shared with SP-B. 

The membrane localization of SP-C presents an 

interesting obstacle to its secretion. This conundrum 

is solved by internalization of the proprotein 

from the limiting membrane of the MVB to small 

lumenal vesicles, an event that is regulated by 

Nedd4-2-mediated mono-ubiquitination of proSP-C 

(20, 58). Fusion of the MVB with a LB leads to SP-Bmediated 

incorporation of SP-C-containing lumenal 

vesicles into existing surfactant membranes that are 

eventually secreted into the airspaces. Thus mature 

SP-C, one of the most hydrophobic peptides known, 

maintains its transmembrane orientation throughout 

biosynthesis. 

Surprisingly, disruption of the SP-C locus in 

mice (Sftpc) has no overt effect on synthesis, trafficking, 

packaging, or secretion of surfactant by 

type II epithelial cells (40, 41). Sftpc / mice survive 

with essentially normal gas exchange apparently 

because SP-B is sufficient for surfactant 

function in vivo. However, SP-C deficiency is associated 

with development of chronic lung disease in 

humans (3) and strain-dependent, progressive lung 

disease in Sftpc / mice. Identification of genes that 

confer strain-dependent sensitivity and/or resistance 

to lung disease in Sftpc / mice remains an important 

and unresolved issue (44). Surfactant films from 

SP-C-deficient mice show minor intrinsic instability 

(40), which may contribute to chronic respiratory 

disease; however, it is equally possible that SP-C has 

an unidentified surfactant-independent function(s). 

Mutations in the SFTPC gene have been linked to 

chronic lung disease, possibly through formation of 

cytotoxic aggregates of mutant SP-C proprotein (for 

review, see Ref. 107). Overall, conservation of SP-C 

among species is consistent with an important function; 

however, despite considerable information 

about SP-C biosynthesis and in vitro surface activity, 

the role of this peptide in surfactant homeostasis and 

lung disease remains obscure. 

Synthesis of Surfactant Lipids 

Phosphatidylcholine 

Phosphatidylcholine (PC) is a major component of 

all cellular membranes (98) and is the most abundant 

surfactant phospholipid (100). The rate limiting 

enzyme for PC synthesis, cholinephosphate 

cytidylyltransferase, is expressed as two isoforms 

(CCT and CCT) encoded by separate genes. Targeted 

disruption of the CCT locus (Pcytla) orthe 

locus encoding another key enzyme in this pathway, 

choline kinase (Chka), results in embryonic 

lethality confirming that PC synthesis is essential 

for early embryogenesis (104, 110). Targeted deletion 

of Pcytla in type II epithelial cells of mice 

resulted in neonatal RDS and death within 10 min 

of birth (94). Lung development in Pcytla / mice 

was normal, but total PC content in the airspaces 

was dramatically reduced and formation of lamellar 

bodies in type II epithelial cells was highly aberrant. 

These results confirm the key role of the 

CCT isoform in synthesis of surfactant PC. 

Approximately half of the PC in surfactant is 

dipalmitoylphosphatidylcholine (DPPC; PC 16:0/ 

16:0), accounting for 40% of total surfactant 

phospholipid (82, 100). DPPC is the lipid considered 

as primarily responsible for the surface tension 

reducing properties of surfactant; therefore, 

regulation of this PC species is likely critical for 

surfactant homeostasis. Two pathways contribute 

to the production of DPPC, de novo synthesis and 

remodeling via lysoPC. The latter pathway accounts 

for up to 75% of DPPC in type II cells and 

involves deacylation of unsaturated PC at the sn-2 

position by a calcium-dependent phospholipase 

A2 (33); peroxiredoxin 6, a calcium-independent 

phospholipase A2, may also contribute to deacylation 

of PC (25, 34). The enzyme responsible for 

re-acylation, lysophosphatidylcholine acyltransferase, 

was recently identified and named LPCATI 

(69). This finding is a significant achievement that 

will facilitate testing of a number of hypotheses in 

vivo. Targeted disruption of the LPCATI locus in 

type II epithelial cells will permit assessment of the 

importance of the remodeling pathway in surfactant 

homeostasis, identification of potential compensatory 

pathways, and the overall importance of 

DPPC for surfactant function in vivo. In addition, 

recent evidence supports the hypothesis that there 

is cross-talk between the remodeling and the de 

novo synthesis pathways (16). Identification of 

transcriptional networks that integrate expression 

of LPCATI and other lipogenic enzymes with surfactant 

proteins should provide important insight 

into the molecular pathways governing surfactant 

homeostasis (FIGURE 1). 

Phosphatidylglycerol 

The second most abundant surfactant phospholipid, 

PG, comprises 10% of the surfactant lipid 

fraction in humans. This anionic phospholipid is 

not a common component of intracellular membranes 

(98). Unlike PC, surfactant PG content and 

molecular species varies significantly among animals 

(82). However, the overall content of anionic phospholipid 

(PGPI) is relatively constant, suggesting 

that the contribution of acidic polyhydroxylated 

phospholipids is more important than the precise 

molecular forms. A key step in PG synthesis is 

conversion of CDP-diacylglycerol to phosphatidylglycerolphosphate 

by phosphatidylglycerophosphate 

synthase (8, 9). Transcription of the gene 

encoding this enzyme (PGS1) is increased threefold 


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during maturation of human lung epithelial cells in 

vitro (103) and is similarly increased in rat type II 

cells when cultured under conditions that promote 

differentiation (65). Targeted disruption of the Pgs1 

locus in type II epithelial cells could provide important 

new information regarding the role of PG 

in LB maturation, surfactant function, and the potential 

for PI to compensate for loss of PG. Although 

the role of PG in surfactant homeostasis 

remains unclear, two recent studies presented 

compelling preliminary evidence that PG can suppress 

viral infection and inflammatory responses 

in the lung (61, 72). These novel findings underscore 

the importance of filling this particular 

knowledge gap. 

Intracellular Trafficking of 

Surfactant Phospholipids 

The fully assembled surfactant lipid-protein complex 

is stored as tightly packed bilayer membranes 

in lamellar bodies of type II epithelial cells. Since 

lamellar bodies lack the requisite enzymes for 

phospholipid biosynthesis (8), surfactant lipids 

must be transported from the site of synthesis in 

the ER to the storage organelle. The identity of 

pathways involved in intracellular trafficking of 

surfactant phospholipids remains one of the major 

unanswered questions in this field. Transport of 

intracellular lipids can occur by at least three pathways, 

including vesicular transport, non-vesicular 

transport, and diffusion at membrane contact sites (98). 

Vesicular transport clearly plays an important 

role in trafficking of newly synthesized surfactant 

proteins via the Golgi to the lamellar body (FIGURE 1). 

Vesicular transport is also important for recycling 

of surfactant components (both proteins and lipids) 

from the alveolar airspaces to the LB via the 

endocytic pathway. The biosynthetic and endocytic 

pathways converge at the MVB/late endosome 

(the boundary between these endosomal 

compartments is not well defined in type II epithelial 

cells). The internal membranes of MVBs are 

enriched in cholesterol and lyso-bisphosphatidic 

acid. Thus the endocytic recycling pathway is likely 

an important source of cholesterol and minor 

phospholipid components of lamellar body membranes 

(FIGURE 1). 

In contrast to vesicular transport of surfactant 

lipids in the endocytic pathway, there is very little 

evidence for anterograde vesicular transport of 

DPPC or PG from the ER to the LB. Ultrastructural, 

autoradiographic analyses of type II epithelial cells 

in vivo detected the appearance of 3 [H]cholinelabeled 

lipids first in the ER followed by the Golgi 

and LB; notably label was not detected in the MVB 

(27). Although these results are consistent with 

trafficking of PC directly to the LB and/or via the 

Golgi, a separate study found that inhibition of 

vesicular transport through the Golgi did not inhibit 

incorporation of newly synthesized lipid into 

the LB (75). A unifying hypothesis posits that PC 

moves directly from the ER to the LB via a nonvesicular 

transport pathway. This hypothesis is supported 

by the localization of the ABC transporter, 

ABCA3, to the limiting membrane of the LB (68, 

111). Disruption of the Abca3 locus results in neonatal 

lethal RDS associated with loss of mature 

lamellar bodies and a dramatic decrease in production 

of lung PG and PC (7, 26, 35, 48). Similar 

findings have been reported for humans with severe, 

recessive ABCA3 mutations (13–15, 32, 39, 66, 

87, 91). Since ABC transporters typically move substrate 

out of the cytosol, it is highly likely that 

ABCA3 is an essential transporter of PC and/or PG 

into the LB. 

How is PC and/or PG transported from the ER to 

ABCA3 in the LB membrane? Intermembrane 

transport of PC and PG is significantly elevated in 

type II epithelial cells relative to whole lung and 

transfer activity increases commensurate with surfactant 

synthesis before birth (38, 63). Intracellular 

phospholipid transport activity was generally attributed 

to phospholipid transfer proteins and to 

PC-TP/StarD2 in particular. However, lung structure 

and function, including LB morphology and 

alveolar DPPC content, was unaffected in Pc-tp / 

mice (97). This outcome was quite unexpected, 

given the specificity of PC-TP/StarD2 for PC (55) 

and its tempo-spatial relationship to surfactant 

synthesis. It is currently not clear whether compensation 

occurs in type II cells of Pc-tp / mice 

or whether PC transfer is normally accomplished 

independently of PC-TP/StarD2. Other members 

of the START domain family (2) could account for 

PC transfer activity in type II epithelial cells, in 

particular StarD10. It is also possible that this function 

could be fulfilled by phospholipid transfer 

proteins lacking a START domain. SCP-2/nsL-TP 

and PI-TP both have PC transfer activity (108), and 

the former is enriched in type II epithelial cells and 

increases during fetal lung maturation (10). Overall, 

the involvement of phospholipid transfer proteins 

in nonvesicular transport of newly synthesized 

phospholipids from the ER to the LB remains an 

important and unresolved question. 

Another possible mechanism for transfer of 

phospholipids between donor and acceptor membranes 

is via diffusion or facilitated exchange at 

membrane contact sites (98) (FIGURE 1). This 

transfer mechanism would likely require the participation 

of accessory proteins to ensure the formation 

of transient contact sites between the 

appropriate donor (ER) and acceptor (LB) organelles. 

It has been suggested that some phospholipid 

transfer proteins could fulfill the role of 



REVIEWS 

both accessory and transfer protein (2). Regardless 

of whether membrane contact is necessary for targeted 

transport of PC or PG from the ER to the LB, 

it is likely that a transfer protein(s) with docking 

motifs specific for each organelle would be required. 

Lamellar Body Maturation 

Based on the foregoing discussion, a simple model 

of lamellar body maturation can be constructed 

(FIGURE 1). The major surfactant phospholipids 

(DPPC, unsaturated PC, and PG) are synthesized in 

the ER. Because there is very little DPPC or PG in 

the ER, newly synthesized phospholipid must be 

rapidly exported from this organelle. Phospholipid 

transport is likely facilitated by specific phospholipid 

transfer proteins containing docking motifs 

that enable cargo loading at the ER and cargo 

discharge at the LB. Although phospholipid transfer 

proteins typically carry only a single cargo molecule, 

transfer can occur very rapidly (5). Import of 

PC and PG into the LB likely occurs through 

ABCA3. An excess of phospholipids at the inner 

leaflet of the LB membrane could drive the assembly 

of internal surfactant membranes (78). Incorporation 

of cholesterol and minor phospholipid 

components into surfactant membranes could be 

mediated by ABCA3, by unidentified lipid transporters 

in the LB membrane, or via the endocytic/ 

recycling pathway. The process leading to highly 

organized, tightly packed surfactant membranes is 

poorly understood but likely requires SP-B and the 

fluidizing effects of cholesterol/minor phospholipids. 

This highly simplified model raises numerous 

questions. Do newly synthesized minor surfactant 

lipid components employ the same transport machinery 

(i.e., phospholipid transfer proteins and 

ABCA3) as PC and PG? Are PC and PG carried by 

the same phospholipid transfer protein or are multiple 

carrier proteins involved? Is phospholipid import 

into the LB accomplished through more than 

one ABC transporter? Are elevated calcium levels in 

the LB required for packing of negatively charged 

(PG-rich) membranes and, if so, what is the molecular 

identity of the calcium pump? How is LB 

size (maturation) monitored/regulated? Proteomic 

analyses of the limiting membrane of the LB, currently 

underway in several laboratories (105), 

should identify candidate proteins that, in turn, may 

provide answers to these and related questions. 

Extracellular Surfactant 

Reorganization of Surfactant Membranes 

in the Alveolar Airspaces 

Transition of the intracellular storage form of surfactant 

(bilayer membranes in LBs) to an extracellular 

functional surface film has been extensively 

studied but is only partly understood (78, 114). The 

alveolar epithelial cell surface is covered by a thin 

aqueous layer (hypophase) in which newly secreted, 

tightly packed surfactant membranes reorganize 

to form a loose network of interconnected 

membranes that contacts the air-liquid interface. It 

is not clear how the structural transformation of 

surfactant membranes is triggered, but potential 

effectors include changes in the hydration of surfactant 

complexes, pH, or calcium concentration 

(29–31, 78, 84, 85). Subsequently, SP-B and SP-C 

are thought to facilitate transfer of surface active 

molecules (particularly phospholipids and, most 

importantly, DPPC) from the membrane network 

to the surface film (see FIGURE 2) (79, 89). Although 

this model of interfacial adsorption is 

widely accepted, translation of the results of in 

vitro analyses to surfactant adsorption in vivo is 

not straightforward. In particular, most in vitro 

studies have used surfactant preparations that are 

significantly diluted (simulating unpacked surfactant 

membranes) and simplified (i.e., simple phospholipid 

mixtures) compared with newly secreted 

surfactant. Moreover, the cellular microenvironment 

(composition and volume of the hypophase) 

may profoundly influence transfer of phospholipids 

from the packed membrane state to the surface 

film (33). In this regard, the results of recent studies 

revealed that packed surfactant membranes 

(similar to newly secreted surfactant) can spontaneously 

adsorb and rapidly transfer surface active 

material into the air-liquid interface, apparently 

without passing through an unpacked state (29, 

47). Thus the mechanisms by which newly secreted 

surfactant phospholipids are transferred to the airliquid 

interface in vivo remains an open question. 

Composition and Function of the 

Surface-Active Film 

Cyclical expansion (inhalation) and contraction 

(exhalation) of alveoli lead to changes in alveolar 

surface area accompanied by altered surface tension. 

Early work by Clements (19a) proposed that 

surface tension must be 2 mN/m at low lung 

volumes to prevent alveolar collapse (atelectasis) 

and rise to a maximum of 20–25 mN/m at higher 

volumes to maintain alveolar stability. This goal is 

achieved by maintenance of an interfacial film 

highly enriched in DPPC, a saturated phospholipid 

that produces extremely low surface tension on 

film compression (1 mN/m). The lack of fluidity 

of pure DPPC films at body temperature is overcome 

by incorporation of phospholipids with unsaturated 

acyl chains and cholesterol into surfactant 

complexes and the interfacial film (10a, 27a, 30a, 

68a). Observation of surfactant films by epifluorescence 

or atomic force microscopy suggests that satu- 


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rated phospholipids (DPPC) segregate into ordered 

micro- and nano-domains, which can be highly 

compressed to produce the requisite low surface tension; 

unsaturated phospholipids and surfactant proteins 

remain in disordered regions that facilitate 

rapid transfer of phospholipids into (and out of) the 

surface film (22a, 27a, 115). Comingling of ordered 

and disordered domains thus provides a 

potential mechanism for maintenance of a stable 

interfacial film. 

Although we have a basic understanding of surfactant 

film dynamics, many details of the underlying 

processes remain unclear. It is assumed that 

DPPC enrichment of the surface film is required to 

support maximal pressures (very low surface tensions) 

at end expiration, but the actual proportion 

of DPPC that reaches the interfacial film is not 

known. Some studies suggested that SP-B and 

SP-C could selectively promote insertion of DPPC 

(99) into the interfacial film, but direct proof for 

this activity is lacking. It has been also proposed 

that compression of the surface film during expiration 

induces progressive refining of the interfacial 

film by squeezing out less stable unsaturated 

species, leading to DPPC enrichment (28, 64, 76). 

Direct examination of the structure and composition 

of the surface film formed from native surfactant 

under physiologically relevant conditions is 

required to confirm or discard these hypotheses. It 

is currently thought that the ability of surfactant to 

produce very low tensions during breathing is dependent 

on formation of a multilayered film at the 

interface (6, 36, 88). However, a comparison of the 

rheological properties of multilayered vs. monolayered 

surfactant films is required to test this model. 

Finally, an active line of research has tried to assign 

specific activities to SP-B and SP-C in remodeling 

of interfacial surfactant films during compression 

and facilitating film re-spreading during expansion 

(see FIGURE 3). Consistent with the neonatal lethal 

phenotype of SP-B-deficient mice, in vitro approaches 

using devices that capture some of the 

physiological compression-expansion constraints 

of the respiratory interface revealed that SP-B is 

required to achieve and sustain the lowest tensions 

during compression. SP-C, on the other hand, may 

cooperate in facilitating interfacial surfactant dynamics, 

including folding of the surface film, maintaining 

the multilayer reservoir attached to the 

interface during compression and promoting efficient 

re-spreading of the film on expansion. Optimal 

performance of surfactant at the interface may 

therefore require cooperation of both hydrophobic 

surfactant proteins (1). Validation of these models in 

vivo requires the development of new genetically 

modified animal models that are not yet available. 

Surfactant Recycling and Surfactant 

Homeostasis 

Compression-expansion cycling leads to progressive 

conversion of the surface active fractions of 


FIGURE 2. Pulmonary surfactant adsorption to the interface and surface film formation 

Processes that may contribute to transport of surface active surfactant species to the interface include 1) direct cooperative 

transfer of surfactant from secreted lamellar body-like particles touching the interface, 2) unraveling of secreted 

lamellar bodies to form intermediate structures such as tubular myelin (TM) or large surfactant layers that have 

the potential to move and transfer large amounts of material to the interface, and 3) rapid movement of surface active 

species through a continuous network of surfactant membranes, a so-called surface phase, connecting secreting 

cells with the interface. 


REVIEWS 

surfactant into much less active lipid/protein 

complexes (43, 95). Inactivation probably includes 

detachment of small particulate entities from the 

interface, changes in lipid/protein organization, 

and oxidation of lipid and protein species on repetitive 

exposure to air (83). Surfactant may also be 

inactivated by incorporation of materials inhaled 

from the upper airways or leaked from capillaries 

(42, 114). Maintenance of a fully functional surfactant 

film therefore requires continual film refinement 

through efficient removal of spent surfactant 

and incorporation of newly secreted complexes. It 

remains unclear whether the distinct surfactant 

structures that coexist in the alveolar pool are specifically 

targeted to functional, recycling, or clearance 

pathways. 

Surfactant homeostasis requires coordinated 

regulation of the processes summarized in 

FIGURE 1. The alveolar surfactant pool is continuously 

depleted through cellular uptake by type 

II epithelial cells and alveolar macrophages as 

well as removal via the mucociliary escalator. It is 

unclear whether cellular uptake of alveolar surfactant 

occurs indiscrimately or whether there is selective 

targeting of specific forms of surfactant to 

type II cells for recycling and/or macrophages for 

degradation. Preliminary support for selective uptake 

of alveolar surfactant by type II cells comes 

from recent studies in SP-D-deficient mice (50, 51). 

Interaction of the collectin SP-D with PI-rich surfactant 

complexes altered surfactant structure and 

enhanced uptake by type II cells but not macrophages. 

Thus, in addition to its well documented 

role in host defense, SP-D plays an important role 

in regulating alveolar pool size. 

The loss of surfactant from the airspaces is balanced 

by secretion of surfactant stored in lamellar 

bodies: regulation of surfactant secretion is therefore 

critical for homeostasis. Several excellent, recent 

reviews provide a comprehensive analysis of 

surfactant secretion and the central role of calcium 

mobilization in this process (4, 29). However, although 

pharmacological and physical stimuli of surfactant 

secretion have been extensively characterized, 


FIGURE 3. Participation of proteins SP-B and SP-C in surfactant dynamics during respiratory compression-expansion 

cycling 

Hydrophobic surfactant proteins play key roles in facilitating optimal dynamic behavior of surfactant during breathing. 

During exhalation, the surfactant surface film folds through three-dimensional transitions, forming a complex structure 

that sustains maximal pressures. Protein SP-C facilitates compression-driven folding of surfactant interfacial films (1), 

expulsion of lipids and lipid/protein complexes from the interface (80, 92), and formation of three-dimensional phases 

(59, 101, 102). SP-B stabilizes the interfacial film (2) and promotes establishment of membrane-membrane contacts 

(22, 24, 86), leading to formation of multilayered membrane arrays (3) (17). SP-B seems to provide mechanical stability 

to compressed films (23), possibly through increasing cohesivity between surfactant layers during maximal compression 

of the surface film. SP-C could promote association of excluded surfactant structures with the maximally 

compressed interface, likely through its NH 2 -terminal segment and/or insertion of palmitoylated cysteines of the protein 

into tightly packed interfacial films (4) (11, 102). Finally, both SP-B (23, 90, 106) and SP-C (73, 74, 81) seem to 

promote insertion (5) and re-spreading (6) of phospholipids from subsurface compartments into the interface during 

expansion. 

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the molecular pathway(s) linking changes in alveolar 

pool size to stimulation of secretion has not been 

identified. 

Depletion of the intracellular pool of surfactant 

via the secretory pathway is balanced in part by 

uptake and recycling of alveolar surfactant in type 

II cells. Recycling is not sufficient to replenish the 

intracellular pool because some internalized surfactant 

is degraded: how internalized surfactant is 

partitioned between the recycling and degradation 

pathways remains an important unresolved question. 

To maintain intracellular pool size, new synthesis 

of surfactant must be coupled to recycling. 

Surfactant pool size also varies significantly with 

age, the alveolar pool being much larger in neonates 

than in adults (18, 50, 113). Overall, maintenance 

of the balance between the intracellular 

storage pool and the functional alveolar pool requires 

tight integration of synthesis, secretion, recycling, 

and degradation. How these pathways are 

modulated in response to quantitative and/or 

qualitative changes in alveolar surfactant remains 

a large and important knowledge gap. 

In summary, although much has been learned 

regarding the composition and function of surfactant, 

virtually nothing is known about the transcriptional 

networks that integrate molecular 

pathways involved in prenatal type II cell maturation, 

surfactant protein and lipid synthesis, and 

alveolar defense. How these molecular networks 

“sense” alveolar pool size in the postnatal lung and 

modulate transcriptional pathways to balance surfactant 

synthesis with surfactant secretion, recycling, 

and degradation is completely unknown. 

Likewise, it is unclear to what extent regulatory 

networks involved in type II cell maturation are 

also involved in alveolar repair and reconstitution 

of surfactant homeostasis following lung injury. 

These critical knowledge gaps are reflected in the 

paucity of new therapies for lung immaturity and 

chronic lung diseases. 

Research in the laboratories of the authors is currently 

supported by grants from Spanish Ministry of Science 

(BIO2009-09694, CSD2007-0010), Community of Madrid 

(S0505/MAT/0283), and Universidad Complutense to J. 

Perez-Gil and The National Heart Lung and Blood Institute 

(HL-056285 and HL-086492) to T. E. Weaver. 

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