Alveolar Surfactant Homeostasis and the Pathogenesis of Pulmonary Disease

Clinical Presentation

Hereditary SP-B deficiency usually presents in full-term infants with unexplained respiratory distress and findings typical of respiratory distress syndrome in preterm infants, the latter lacking surfactant on a maturational basis (21). Infants may have a family history of unexplained neonatal death or consanguinity. Cyanosis and respiratory distress are usually noted in the first hours after birth, followed by respiratory failure that is refractory to surfactant replacement, ventilation, and other supportive care. Initial chest X-rays show diffuse opacification consistent with atelectasis. Respiratory failure is progressive, generally leading to death within weeks to months of age despite assisted ventilation, oxygen, or extracorporeal membrane oxygenation.

Pathogenesis and Diagnosis

The SFTPB gene is located on chromosome 2 (OMIM #178640). Hereditary SP-B deficiency has been linked to a number of distinct mutations in the SFTPB gene. These include deletion, termination, missense, and substitution mutations that interfere with the synthesis of proSP-B or produce an aberrant proSP-B protein that is not fully processed, leading to a loss of the active, mature SP-B peptide. Mature SP-B is a 79-amino-acid, amphipathic peptide that interacts closely with the surface of phosphatidylcholine-rich membranes in surfactant. The active peptide is produced by proteolytic processing of a 381-amino-acid proprotein during trafficking from the endoplasmic reticulum (ER) to multivesicular and lamellar bodies (2). A relatively common insertional mutation, 121ins2, causes the synthesis of an unstable mRNA with loss of synthesis of both proSP-B and SP-B peptides (9, 21). Neither proSP-B nor SP-B is detected in the lungs of patients bearing the 121ins2 mutation. In contrast, other SFTPB mutations result in the production of an incompletely processed proSP-B protein that is detected within the type II cells.

In most forms of hereditary SP-B deficiency, the active SP-B peptide is absent in the surfactant isolated from alveolar wash or in the alveoli as assessed by immunohistochemistry (22). Consistent with the importance of SP-B in surfactant function, surfactant activity is deficient in material isolated from patients with this disorder. Histological findings in hereditary SP-B deficiency include (a) the accumulation of eosinophilic or periodic acid-Schiff (PAS)–positive, lipoproteinaceous material in the alveolar spaces, which often contain foamy alveolar macrophages; (b) hyperplastic alveolar epithelia with prominent type II cells; and (c) thickening of the alveolar septa, which is caused by proliferation of interstitial fibroblasts (Figure 2). At the ultrastructural level, type II cells lack lamellar bodies and contain large, atypical, multivesicular bodies, likely representing the failure of phospholipid membrane fusion during the packaging of surfactant lipids (Figure 2). Tubular myelin is absent, and abnormally processed proSP-C and SP-A accumulate with lipids in the alveolar spaces, providing a basis for the pathological diagnosis of congenital alveolar proteinosis. The alveoli are generally remodeled and lined by cuboidal type II cells, although this varies depending on the age of the infant and supportive care. Inflammation is also variable, generally consisting of foamy alveolar macrophages. Physiological, biochemical, and histological findings in lungs of patients with hereditary SP-B deficiency are consistent with those seen in Sftpb^−/− gene targeted mice, supporting the concept that the pathogenesis of lung dysfunction in this disorder is related primarily to the lack of SP-B in type II cells and in the alveolus (23).

The diagnosis of hereditary SP-B deficiency, when suspected clinically and pathologically, is confirmed by the identification of mutations in the SFTPB gene. Because SP-B is required for both intracellular and extracellular aspects of surfactant homeostasis, the disease has not been treated successfully with surfactant replacement and supportive care. Survival has been extended by lung transplantation in a small number of infants with hereditary SP-B deficiency (24).

Clinical Presentation

Most infants with mutations in the ATP-binding cassette, subfamily A, member 3 (or ABCA3) gene (OMIM #610921) present with severe respiratory distress and cyanosis requiring ventilatory support in the immediate newborn period. The clinical findings are consistent with surfactant deficiency (25). Radiographic findings demonstrate diffuse alveolar disease and atelectasis. Respiratory failure occurs despite surfactant replacement and ventilatory support, and most infants die in the first months of life.

Pathogenesis and Diagnosis

The ABCA3 gene is located on chromosome 16 (OMIM #601615). ABCA3 deficiency is inherited as an autosomal recessive mutation, and >150 distinct mutations have been identified in association with severe neonatal lung disease. The vast majority of ABCA3 mutations cause neonatal lung disease and death within the first months of life. Less frequently, milder ABCA3 mutations (for example, E292V) are associated with chronic lung disease with features of ILD, desquamative interstitial pneumonitis, and nonspecific interstitial pneumonitis presenting in childhood (26, 27). The ABCA3 gene encodes a large, multiple-membrane-spanning polypeptide that is a member of the family of ABC transporters, which includes the cystic fibrosis transmembrane conductance regulator (CFTR) and the multiple drug resistant transporter (MDR). Although the ABCA3 protein is expressed in many tissues, it is expressed highly in type II cells, where it is located at the limiting membranes of lamellar bodies (28, 29).

ABCA3 mutations are associated with histological features of congenital alveolar proteinosis (25, 27). The respiratory epithelium undergoes cuboidal hyperplasia with remodeling and loss of alveoli, as well as accumulation of lipid-rich macrophages in the airspaces (Figure 2). Surfactant from patients with ABCA3-related lung disease is deficient in both PG and PC and has little surface activity (30). ABCA3 is located at the limiting membrane of lamellar bodies in alveolar type II cells, where it plays an important role in lipid transport, including both cholesterol and PC (29, 31). At the ultrastructural level, normal lamellar bodies are lacking in lungs of patients with ABCA3-related disease (25, 27). The presence of small intracellular organelles containing electron-dense material, likely representing lamellar bodies that lack appropriate lipid constituents, is useful in the tentative diagnosis of ABCA3-related disease (Figure 2). In general, the surfactant proteins A, B, C, and D are detected by immunohistochemistry in lung tissue from patients with lung disease caused by mutations in ABCA3. As in hereditary SP-B deficiency, the diagnosis of ABCA3-related disease is made by clinical and pathological findings supported by histological and ultrastructural studies. Definitive diagnosis is made by the identification of mutations in the ABCA3 gene locus. ABCA3-related lung disease is severe and usually results in death in the first months of life. Life has been extended by lung transplantation in a number of patients with ABCA3 mutations (27, 30).

Clinical Presentation

The index case was an infant who developed respiratory symptoms at six weeks of age and was diagnosed subsequently with interstitial pneumonitis (32). DNA sequence analysis of the SFTPC gene identified a mutation in a donor splice site (c.460 + lG → A) of one allele that resulted in deletion of exon 4 and consequent loss of 37 amino acids in the C-terminal peptide of the proprotein (referred to as SP-C^Δexon4). A separate SFTPC mutation (exon 5+128T→A) that results in substitution of glutamine for leucine (L188Q) was recently identified in two independent kindreds (33, 34). Family members exhibited variable onset of disease, from infancy to adulthood, with autosomal dominant inheritance and incomplete penetrance.

Pathogenesis and Diagnosis

The SFTPC gene is located in chromosome 8 (OMIM #178620). To date, approximately 40 missense, splice, or frameshift SFTPC mutations have been associated with familial or sporadic ILD. In general, SFTPC mutations are located in the brichos domain of proSPC, causing misfolding or aberrant processing of the precursor. Histological features of lung disease caused by mutations in the SFTPC gene include (a) diffuse alveolar damage of varying severity, (b) interstitial thickening with mild lymphocytic inflammation, (c) thickening of the alveolar septa, (d) accumulation of foamy alveolar macrophages and variable amounts of eosinophilic alveolar proteinosis material, and (e) regenerating alveolar epithelium lined by hyperplastic type II cells (Figure 2).

Mutations that alter the ability of a protein to fold correctly result in retention of the unfolded/ misfolded protein in the ER(35, 36). Accumulation of unfolded or misfolded protein in the ER, a condition known as ER stress, induces an ER-to-nucleus signal pathway called the unfolded protein response, which increases transcription of chaperones that promote protein folding. In addition, translation of mRNA is attenuated, effectively slowing entry of newly synthesized protein into the ER and increasing the ratio of chaperone to unfolded protein. Proteins that fail to fold under these conditions are diverted to the ER-associated degradation (ERAD) pathway, in which misfolded protein is translocated to the cytosol for degradation by 26S proteasome. Failure to eliminate rapidly the terminally misfolded proteins can lead to formation of cytotoxic aggregates and/or trigger cell death pathways. Both SP-C^Δexon4 and SP-C^L188Q proproteins cause ER stress and readily aggregate when the ERAD pathway is compromised (37, 38).

Targeted expression of SP-C^Δexon4 in type II cells of transgenic mice resulted in dose-dependent cytotoxicity leading to disruption of lung morphogenesis and respiratory failure at birth (39). Importantly, lung dysmorphogenesis occurred in the presence of two wild-type Sftpc alleles, consistent with a toxic gain-of-function effect. This phenotype was not due to degradation and loss of mature SP-C peptide in the airspaces, since perinatal lung development and function were normal in Sftpc^−/− mice (40). Collectively, these results suggest that certain SFTPC mutations lead to irreversible misfolding of the SP-C proprotein and accumulation of misfolded protein that, in turn, leads to cell injury and death. This hypothesis is supported by the results of experiments confirming the cytotoxicity of mutant SP-C protein in cell culture (41, 42).

The cytotoxic effect of mutant SP-C fits well with the emerging concept that epithelial cell injury and aberrant re-epithelialization are critical steps in the pathogenesis of ILD (43). What remains unclear is why the onset and severity of disease are so variable within a kindred. For example, in the L188Q kindred, the age of onset ranged from 4 months to 57 years, with histopathological findings ranging from non-specific interstitial pneumonitis in infants to usual interstitial pneumonitis in older patients (33). Phenotypic variability may be due in part to the influence of other genes, which render some patients more susceptible or resistant to disease, and in part to environmental stresses that may trigger or exacerbate disease. Interestingly, cells that stably expressed SP-C^Δexon4 in culture adapted to chronic ER stress imposed by the mutant protein and grew normally; however, adaptation was accompanied by greatly increased susceptibility to viral-induced cell death (42). These findings provide a potential explanation for delayed onset of disease but have yet to be translated to an animal model.

Two recent studies of IPF patients support a role for ER stress in pathogenesis. One study detected markers of ER stress and apoptosis in alveolar epithelial cells of IPF patients but not in those of patients with chronic obstructive pulmonary disease (44). The other study confirmed evidence of both ER stress and herpesvirus infection (45). Thus, ER stress may be a molecular pathway common to both familial and idiopathic forms of ILD. The precise roles of ER stress and environmental stress in the initiation and/or progression of ILD remain to be defined.

Clinical Presentation

PAP is rarely diagnosed in childhood; it more commonly presents in adulthood in both acquired and secondary forms. Patients usually present with progressive dyspnea, exercise intolerance, and clubbing.

Radiologic studies demonstrate diffuse alveolar opacities, termed “crazy-paving,” but lack the fibrosis or remodeling associated with ILD (49). Alveolar wash or histologic assessment of lung biopsy demonstrates the accumulation of surfactant lipids and proteins and foamy alveolar macrophages in the lung. Surfactant lipids and proteins, including SP-A, SP-B, SP-C, and SP-D, accumulate in large quantities, and the material isolated from PAP patients maintains its surface-active properties. Thus, lung function and structure are generally well preserved. The pathological findings in acquired PAP are distinct from those in genetic pulmonary disorders related to SP-B, SP-C, or ABCA3, wherein surfactant is dysfunctional with resultant respiratory failure, pulmonary fibrosis and remodeling.

Pathogenesis and Diagnosis: Role of GM-CSF in Acquired PAP

The critical role of GM-CSF signaling in the pathogenesis of PAP was first recognized from studies in transgenic mice lacking GM-CSF (50, 51) or the common β-chain of the GM-CSF receptor (52). Both of these mouse models developed severe PAP with features virtually identical to those in human patients with acquired PAP. The mouse models demonstrated that the disorder was related to the inability of alveolar macrophages to clear or metabolize surfactant lipids and proteins. Replacement of GM-CSF in the lung rapidly restored alveolar macrophage differentiation and surfactant clearance, correcting the PAP (53, 54). The recognition that most adult patients with acquired PAP have high circulating levels of anti-GM-CSF autoantibodies provided a critical link between the mouse studies and the clinical disorder (55). Thus, it has been established that GM-CSF plays a critical, nonredundant role in the regulation of differentiated functions of the alveolar macrophage in the lung. Its absence results in failure to degrade surfactant components, leading to the progressive accumulation of normal lipids and proteins in the lung (56, 57). Because alveoli are well preserved in PAP, lung function can be restored by physical removal of the excess surfactant using alveolar wash or by treatment with GM-CSF using lung aerosol or systemic administration.

Role of GM-CSF Receptors in Hereditary PAP

GM-CSF activity requires signaling via the dimerization of its receptors, consisting of an α-chain and common β-chain that activate JAK/STAT5 signaling to enhance macrophage differentiation and function. Dysfunction of the common β-chain of the GM-CSF receptor (OMIM #300770) has been associated with PAP (58). Recently, mutations in the α-chain of the GM-CSF receptor (OMIM #306250) were implicated in the pathogenesis of PAP (14, 15). A number of patients with PAP related to mutations in the α-chain of the GM-CSF receptor have now been identified, usually presenting with signs and symptoms in childhood. Clinical and histopathological features are similar to those of acquired PAP, although these patients usually present at earlier ages (Figure 2).

The diagnosis of hereditary PAP is supported by clinical, pathological, and radiological findings. The definitive diagnosis can now be made by the identification of mutations in the genes encoding the GM-CSF receptors. Since some of the GM-CSF α-chain mutations retain residual activity, it is expected that some patients will respond to treatment with additional GM-CSF. PAP caused by mutation in the common β-chain in mice was rescued by bone marrow transplantation (59), providing support for the feasibility of future cell-based therapies for refractory PAP in patients with defects in GM-CSF signaling.