Table of Contents  

Whitsett: The molecular basis of pulmonary surfactant – lessons from newborn infants

Introduction

It is difficult to write in the first person regarding work carried out in our laboratories within the Division of Pulmonary Biology at Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine from 1980 to the present. I will summarize research that begins in 1980 and continues to the present. During this time, more than 60 post-doctoral coworkers, 15 graduate students, and countless collaborators, both here in Cincinnati and worldwide, have made important contributions to our published and ongoing work. The scope and complexity of the work has been enabled by sequential technical advances in molecular biology during this time period, which have provided tools and scientific approaches that were unconceivable prior to 1980. Such technology continues to advance at seemingly exponential rates. I have been most fortunate to find trainees, coworkers, and collaborators without whom there would have been little progress in this work. We have worked in a period during which the mysteries of cell and molecular biology have unfolded in a most remarkable way. Our work began, with the frustration that many paediatricians shared, in the treatment of preterm infants with respiratory distress, which prior to the 1970s was almost invariably fatal. Our work led to the identification and cloning of the surfactant proteins and genes that, in turn, provided the molecular ‘tool kit’ with which to explore intriguing aspects of lung biology. Our findings supported the approval of mammalian-based surfactant replacements by the US Food and Drug Administration that have transformed respiratory management of premature infants in the neonatal intensive care units worldwide.

The link between pulmonary surfactant and idiopathic respiratory distress syndrome

Hindsight, with its visual and conceptual acuity, provides the unified framework to understand respiratory distress syndrome (RDS), a common, life-threatening condition associated with premature birth. As late as the 1960s, the causes of this ‘idiopathic’ disorder were unclear and were ascribed variously to pulmonary ischaemia, to neonatal heart failure, and to surfactant deficiency.13 The critical importance of surfactant deficiency in the pathophysiology of the disease was identified by seminal work of Mead and Avery3 and, later, by the finding that organic solvent extracts of animal surfactants restored lung function in preterm animals and infants. Primary work from the laboratories of Drs B Robertson, F Adams, T Fujiwara, and J Clements,47 and others supported the utility of surfactant lipid extracts or surfactant lipid mixtures for replacement therapy of RDS that has now become standard treatment. Pioneering work from the Clements laboratory clarified the importance of lipid films formed by pulmonary surfactant lipids that reduce surface tension, that ultimately led to the development of a fully synthetic lipid surfactant.8 By the early 1980s, phospholipids were thought to be sufficient to explain much of the surface activity in organic solvent extracts of surfactant. Lacking, however, was a clear explanation for the remarkable physical properties of pulmonary surfactants that differ from purified lipids alone. Surfactant lipids spread virtually instantaneously and provide stability at alveolar surfaces in spite of dynamic compression and re-expansion of the lung, and are able to reduce surface tension in the presence of serum and other proteins that inhibit surface activity.

Surfactant proteins play a critical role in surface tension reduction and in alveolar homeostasis

As the phase transition temperature of the most abundant lipid in surfactant (dipalmitoyl-phosphatidylcholine) is higher than normal body temperature, pure lipids are in a gel or solid phase rather than in a fluid phase at normal body temperature, and therefore are unable to spread rapidly or provide the full properties of mammalian surfactants. These observations provided the need to search for the unique proteins, lipids or other constituents that conferred this remarkable surface activity on lung surfactants. Subsequent studies in our laboratory and others led to the identification of small, hydrophobic surfactant proteins, now termed SP-B and SP-C, that confer surface activity.913 Our work also clarified the important aspects of structure and function of surfactant proteins A, B, C and D that were subsequently found to have distinct roles in surfactant homeostasis and innate immunity. Many laboratories worldwide have made important contributions to our present understanding of pulmonary surfactant. In this review, I will summarize our work on ‘surfactant homeostasis’ and its impact on the unanticipated discoveries related to molecular and cellular biology of the lung.

Isolation, identification and cloning of the surfactant proteins and related genes

Our initial research goals were focused on specifically identifying the low levels of non-serum proteins present in pulmonary surfactant and in the organic solvent extracts of lung surfactants that had been recently developed for therapy of RDS. By carefully dialysing the lipid fractions of whole-lung surfactant in chloroform/methanol, two small-molecular-weight, lipid-associated proteins were isolated and shown to be novel proteins unrelated to previously known serum or lung proteins9,10 (Figure 1). These two hydrophobic proteins were identified as distinct peptides (now known to be SP-B and SP-C) that when added to purified lipid mixtures, conferred full surfactant-like activity.1113 SP-B and SP-C were identified in surfactant lipid extracts developed for surfactant replacement therapies for infants, including cow and calf lung surfactant extracts. With antibodies and partial peptide sequences from human and cow surfactants we cloned two novel genes [now termed SFTPB (encoding surfactant pulmonary-associated protein B) and SFTPC (encoding surfactant pulmonary-associated protein C)] and that provided the nucleotide sequences encoding the proteins. The genes and proteins were found to be distinct from other surfactant proteins, including SFTPA (surfactant pulmonary-associated protein A; SP-A) and SFTPD (surfactant pulmonary-associated protein D; SP-D).1418 We isolated human and mouse cDNAs and the genomic loci of the genes were determined and sequenced.1922 These discoveries provided the ability to synthesize recombinant SP-A, SP-B, SP-C and SP-D, and to generate useful antibodies to isolate the regions of the genes that conferred the unique tissue-specific expression of the surfactant proteins in the respiratory epithelium. Antibodies produced against each protein enabled the study of their synthesis, processing, secretion and metabolism in lung cells. Knowing the structures of SP-A and SP-D and the genes encoding them, we identified unique functions of the surfactant proteins SP-A, SP-B, SP-C and SP-D in surfactant homeostasis (Figure 2).2224 The antibodies and protein sequences for bovine and human surfactant proteins allowed us to assess the potential immunological consequences of administering animal-based surfactant preparations to preterm babies.25 Surfactant proteins B and C were evolutionarily highly conserved and relatively non-immunogenic. Preterm babies did not produce detectable antibodies against bovine surfactants given to them for treatment of RDS in clinical studies.25

FIGURE 1

Discovery of SP-B and SP-C in lipid extracts of pulmonary surfactant. The abundant lipids and remarkable hydrophobicity of surfactant proteins SP-B and SP-C, which constitute only a small fraction of the composition of surfactant, complicated their isolation and identification using standard protein methodologies. Prolonged dialysis of the lipid-rich surfactant fraction in chloroform–methanol was used to remove the lipids, leading to the isolation of the hydrophobic proteins, SP-B and SP-C. Antibodies and partial-peptide sequences generated against the proteins were used to clone the unique genes encoding them.

5-3-3-fig1.jpg
FIGURE 2

Regulation of surfactant homeostasis in the alveolus. Surfactant homeostasis is controlled at multiple levels, including (1) transcription of genes controlling surfactant lipid and protein production in type II epithelial cells; (2) surfactant lipid and protein synthesis processing, routing, and storage in lamellar bodies or secretory vesicles; (3) secretion of lipids and proteins into the alveoli; (4) assembly of alveolar surfactant forms into large aggregates, e.g. tubular myelin and multilayers; (5) conversion and reuptake of small aggregates by type II epithelial cells; and (6) the uptake and catabolism of surfactant remnants by alveolar macrophages, a process requiring GM-CSF. Surfactant proteins play critical and distinct roles in the packaging, assembly, organization, function and fate of alveolar surfactant.

5-3-3-fig2.jpg

Transgenic mice to elucidate the functions of the surfactant proteins in vivo

Although in vitro studies of SP-A, SP-B, SP-C and SP-D provided initial insights into their likely roles in vivo, we used advances in genetics and cell biology to produce transgenic mice in which genes were deleted, added or mutated, a technology that has revolutionized mammalian biology in recent decades. Cloning and sequencing of the mouse genes encoding the surfactant proteins made possible ‘gene targeting’ of each genomic locus to provide some clarity to their roles in lung biology.2632 Table 1 and Figures 2 and 3 summarize findings from the gene targeting of the surfactant proteins in our laboratories. Figure 2 provides a schema of the synthesis, trafficking and functions of the surfactant proteins, their production by the alveolar type II cells that produce the great majority of surfactant lipids and proteins in the lung. The surfactant proteins and their interactions with lung phospholipids play critical roles in (1) surface tension reduction, (2) innate immunity via their interactions with lipopolysaccharides and pathogens and (3) inflammation.23 SP-A and SP-D, the pulmonary collectins, are C-type lectins that bind complex carbohydrate surfaces of particles and pathogens that play an important role in innate immunity of the lung, mediating clearance and inflammatory responses to viruses and bacterial and fungal pathogens.3335 SP-A and SP-D also have distinct roles in the structural organization of the various lipid forms present in the alveolus, influencing tubular myelin formation and surfactant metabolism, respectively.30,31 SP-A is required for the production of tubular myelin, a unique, highly organized form of surfactant lipids, whereas SP-D is critical to the regulation of endogenous lung lipid pool sizes and in the suppression of oxidant-related lung inflammation. SP-D gene-targeted mice develop spontaneous pulmonary lipidosis, inflammation and emphysema.36 SP-B and SP-C are critical for the spread and stability of surfactant lipids and influence the packaging and metabolism of intracellular and extracellular surfactant.23,24

TABLE 1

Gene targeting in transgenic mice demonstrates the functions of surfactant-associated proteins

Surfactant protein Result from gene targeting
ABCA3 Neonatal respiratory failure, ILD
SP-B Neonatal respiratory failure, ILD
SP-C Pulmonary fibrosis, susceptibility to pulmonary infections, ILD
TTF-1 Neonatal RDS, pulmonary dysplasia and hypothyroidism
GM-CSF Pulmonary alveolar proteinosis
SP-A Innate immune deficits, lack of tubular myelin
SP-D Emphysema, lipidosis and innate immune deficits

ABCA3, ATP-binding cassette subfamily A member 3; GM-CSF, granulocyte–macrophage colony-stimulating factor; ILD, interstitital lung disease; TTF-1, thyroid transcription factor 1.

FIGURE 3

Lung histology in transgenic mice bearing mutations and deletions in surfactant-associated genes. Lung histology from transgenic mice in which mutations or deletions in genes critical for lung formation, synthesis and lipid homeostasis recapitulate findings in patients. Shown is lung histology from normal newborn mice and from Abca3–/–, Ttf1PM mutant, heterozygous SP-B knockout mice exposed to hyperoxia, SP-C–/– and granulocyte–macrophage colony-stimulating factor (GM-CSF)–/– gene-targeted mice. Pathological findings associated with the mutations in genes regulating surfactant homeostasis and function closely phenocopy findings in patients bearing mutations in the genes. Arrows indicate pulmonary fibrosis in adult SP-C–/– mice.

5-3-3-fig3.jpg

The gene promoters from surfactant-related proteins provided new tools for study of lung development and disease

Our discovery that the surfactant proteins and Clara cell secretory proteins (CCSPs) were highly restricted to the respiratory epithelial cells of the lung provided the opportunity to identify the molecular determinants of lung-specific gene expression that were previously unknown. As in other tissues, organ-specific and developmental regulation of gene expression is mediated by the precise temporal and spatial signalling that, in turn, informs the activities of transcription factors that control gene expression. We explored the regulatory sequences in the 5 primef-lanking regions of each of the surfactant proteins and the CCSP gene, demonstrating that each required the activity of TTF-1 (thyroid transcription factor 1, also known as NKX2–137) that had previously been shown to regulate a number of thyroid-specific genes. The critical role of TTF-1, a homeodomain-containing transcription factor of the NKX family of proteins, was demonstrated in studies that continue to the present.3742 TTF-1 had important but diverse roles in lung morphogenesis, perinatal lung maturation, mucous cell differentiation and oncogenesis. We found that TTF-1 functioned in a transcriptional network with a number of other nuclear transcription proteins, including FOX (Forkhead box homologue family members), GATA-6, CEBP-α (CCAAT–enhancerbinding protein alpha), NFATc3 (nuclear factor of activated T-cells, cytoplasmic 3) and others, that regulate lung gene expression critical for lung formation, function and repair. These findings provided foundations for understanding lung morphogenesis, perinatal maturation and other aspects of respiratory epithelial biology.43

Remarkably, and perhaps fortuitously, relatively small regulatory sequences from SP-C and the CCSP genes [SFTPC and SCGB1A1 (secretoglobin, family 1A, member 1), respectively] provided all of the genetic information needed to express genes selectively in respiratory epithelial cells of transgenic mice. We developed the sequences and genetic tools to delete, mutate or add genetic information to lung cells during development and thereafter.4448 Analysis of the regulatory cassettes present in the SP-C (SFTPC) and CCSP (SCGB1A1) genes has been highly useful for the production of transgenic mice used to model and treat respiratory disorders. We were able to target genes throughout the respiratory epithelium or, more selectively, to target alveolar or conducting airway epithelial cells. Under conditional regulation of doxycycline, this system has been utilized by hundreds of laboratories for the study of lung morphogenesis, repair, oncogenesis and inflammation.4950 As TTF-1 was an excellent marker for human lung adenocarcinomas, we developed a monoclonal antibody against TTF-1 that has been widely used for pathological diagnoses of lung cancer throughout the world.5152 With the advent of mRNA microarray analysis, we identified the genes and networks of genes regulated by TTF-1, critical for lung morphogenesis, maturation and other respiratory epithelial functions at the genome-wide level.53 The application of this powerful technology, combined with advances in informatics and systems biology, has provided novel insights into the genetic mechanisms regulating surfactant production and lung maturation. mRNA microarray analyses of neonatal mice with mutations in TTF-1 were used to identify a number of genes critical for lung formation and repair, including SOX2 [also known as SRY (sex-determining region Y)-box 2],5455 required for airway cell differentiation and proliferation, and SPDEF (Sam pointed domain-containing Ets-like factor),5657 the latter required for mucous cell metaplasia associated with asthma, CF (cystic fibrosis) and chronic obstructive pulmonary disease (COPD). We identified an important role for lung epithelial cell transcription factors in innate immunity and inflammation by mRNA microarray analyses, findings that are highly relevant to the pathogenesis of common chronic lung diseases.9,56 Using rapidly evolving bioinformatic approaches, novel networks and pathways synchronizing gestational age and surfactant synthesis and other aspects of lung maturation were identified.5859

Discovery of the ‘surfactopathies’ and interstitial lung diseases caused by mutations in the surfactant proteins

The genes and antibodies used for study of the surfactant proteins proved highly useful for the study of human lung development, lung cancer, and a number of idiopathic lung diseases, including severe neonatal respiratory failure in full-term infants.23,24 These studies were primarily led by Dr Lawrence Nogee and coworkers, and represent a long-standing collaboration between our laboratory and others interested in the study of neonatal lung disease. Mutations in genes encoding SP-B, SP-C and ABCA3 (a lipid transporter expressed in alveolar type II cells) were linked to respiratory failure in newborn and older children.6068 The severe abnormalities in lung function and structure seen in these infants were remarkably similar to findings in transgenic knockout mice produced for SP-B, SP-C and ABCA3, findings that have provided clarity into the pathogenesis of these severe lung disorders (Figure 2). Antibodies against each protein have been useful for the diagnosis of, and understanding the cellular processes underlying, lung dysfunction, epithelial cell injury and lung remodelling that accompany the genetic disorders of surfactant synthesis. Identification of specific mutations in the surfactant-associated protein genes and ABCA3 have led to new genetic diagnostics needed for clinical decision-making and for genetic counselling that is now available for these ‘surfactopathies’.

Gene targeting of granulocyte–macrophage colony-stimulating factor and related receptors in mice provide insight into the pathogenesis and treatment of idiopathic pulmonary alveolar proteinosis

The important role of granulocyte–macrophage colony-stimulating factor (GM-CSF) in haematopoietic development was well established when gene targeting of both GM-CSF and the GM-CSF receptor (GM-CSFR; common β-chain) resulted in the unexpected finding that, as GM-CSF–/– and GM-CSFR –/– mice had minimal abnormalities in haematopoiesis, both developed severe pulmonary alveolar proteinosis (PAP) with features identical to those seen in adult patients with PAP.6970

In PAP, the lungs fill with surfactant lipids and proteins, leading to life-threatening respiratory compromise, with treatment comprising lung lavage. In a series of studies with many co-investigators (in mice, primates and humans), we showed that GM-CSF signalling was required for alveolar macrophages to catabolize surfactant lipids and proteins.71 Provision of GM-CSF to the lungs of GM-CSF-deficient mice, by inhalation or genetic engineering, reversed the alveolar proteinosis.72,73 Likewise, transfer of bone marrow from normal GM-CSFR-sufficient mice to GM-CSF R-deficient mice reversed PAP, setting the stage for the development of new treatments for the disease in humans.70 These studies clarified the important role of the alveolar macrophage in surfactant catabolism.71,74 GM-CSF signalling regulated PU.1, a transcription factor mediating innate immune functions and surfactant clearance in alveolar macrophages.75 Clinical studies are ongoing to utilize GM-CSF or cell and gene transfer for correction of PAP in children and adults.

Biostatement

My research began in the premolecular era and it was not possible to fully anticipate how rapidly advances in biotechnology, cell and molecular biology, genetics and informatics would fuel the identification of the surfactant proteins and their importance in lung function and human disease. The tools and insights gained from the study of newborn infants provided the basis of present-day models of alveolar homeostasis (Figure 2). Surfactant homeostasis is maintained by (1) regulation of gene transcription; (2) lipid and protein synthesis, trafficking and packaging of surfactant proteins and lipids in type II alveolar cells; (3) interactions of surfactant lipids and proteins that produces the unique structural forms of alveolar surfactant that reduce surface tension, preventing atelectasis; and (4) reuptake and/or catabolism of surfactant lipids and proteins by the alveolar epithelium and alveolar macrophages. Our studies of alveolar surfactant provided the tools to uncover processes controlling lung morphogenesis, differentiation, innate immunity and repair that have generated new insights into the pathogenesis of pulmonary diseases. Surprisingly, the study of RDS in premature newborn infants has become the scientific foundation for understanding the pathogenesis and treatment of lung diseases affecting children and adults.

Acknowledgements

The work has been supported by the National Institutes of Health, Heart, Lung and Blood Institute, the March of Dimes, and by trainees who were supported by the Parker B Francis Foundation and the American Lung Association. The Research Foundation of the Cincinnati Children's Hospital Medical Center (CCHMC) and the University of Cincinnati College of Medicine made ongoing contributions to the development of the programme in the Division of Pulmonary Biology. Past and present colleagues and trainees in the Division of Pulmonary Biology at CCHMC, including Drs Timothy E Weaver, Thomas R Korfhagen, Stephan W Glasser, Bruce C Trapnell, Machiko Ikegami, Susan E Wert, Alan H Jobe, Lawrence Nogee, Yan Xu, Kathryn A Wikenheiser-Brokamp, Anne-Karina T Perl, Robert J Bohinski and many others, made seminal contributions to the work described herein. Mr William Hull, Ms Jean Clark, Mr Michael Bruno and Mr Michael Burhans provided truly outstanding support during the early years of the research in surfactant biology. Ms Ann Maher and Ms Cindy Wilson have provided support for all of us in the Division of Neonatology, Perinatal and Pulmonary Biology at CCHMC. Throughout our studies, CCHMC has provided an outstanding academic, clinical and research environment in which to conduct our studies.

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