The molecular basis of pulmonary alveolar proteinosis

Abstract

Pulmonary alveolar proteinosis (PAP) comprises a heterogenous group of diseases characterized by abnormal surfactant accumulation resulting in respiratory insufficiency, and defects in alveolar macrophage- and neutrophil-mediated host defense. Basic, clinical and translational research over the past two decades have raised PAP from obscurity, identifying the molecular pathogenesis in over 90% of cases as a spectrum of diseases involving the disruption of GM-CSF signaling. Autoimmune PAP represents the vast majority of cases and is caused by neutralizing GM-CSF autoantibodies. Genetic mutations that disrupt GM-CSF receptor signaling comprise a rare form of hereditary PAP. In both autoimmune and hereditary PAP, loss of GM-CSF signaling blocks the terminal differentiation of alveolar macrophages in the lungs impairing the ability of alveolar macrophages to catabolize surfactant and to perform many host defense functions. Secondary PAP occurs in a variety of clinical diseases that presumedly cause the syndrome by reducing the numbers or functions of alveolar macrophages, thereby impairing alveolar macrophage-mediated pulmonary surfactant clearance. A similar phenotype occurs in mice deficient in the production of GM-CSF or GM-CSF receptors. PAP and related research has uncovered a critical and emerging role for GM-CSF in the regulation of pulmonary surfactant homeostasis, lung host defense, and systemic immunity.

Figure 1. Schematic representation of surfactant homeostasis and regulation by GM-CSF

Surfactant proteins A–D are synthesized in alveolar epithelial cells type II (AEC-II) (A) processed as they are transported through the endoplasmic reticulum and Golgi apparatus (B) into lamellar bodies (SP-B, SP-C) or secretory vesicles (SP-A, SP-D). Surfactant lipids are also synthesized in AEC-II and transported into lamellar bodies in part via the membrane lipid transporter ABCA3. Mature surfactant is secreted into the alveolar space via lamellar bodies and secretory vesicles (SV) where they form large surfactant aggregates, including tubular myelin (C). Tubular myelin comprises tightly packed ‘leaflets’ of surfactant that ‘unravel’ providing a source of surfactant phospholipids and proteins (D) forming surfactant mono- and multi-layers that reduce alveolar surface tension and prevent alveolar collapse. Spent surfactant expelled from the monolayer as small surfactant aggregates (E) and approximately 70% is internalized by AEC-II (F) and 30% is internalized by alveolar macrophages (G). In AEC-II, half is recycled and half is catabolized. In alveolar macrophages, surfactant is translocated in endosomes that fuse with lysosomes (L) to form phagolysosomes where it is catabolized (H). Catabolism of surfactant lipids and proteins in alveolar macrophages requires stimulation by GM-CSF (I) via the transcription factor PU.1 by a mechanism that has not been defined.

Figure 2. Disorders of homeostasis

Surfactant homeostasis is achieved by the balanced production of surfactant in AEC-II and its recycling or catabolism in AEC-II and catabolism in alveolar macrophages. The PAP syndrome, characterized by accumulation of surfactant, can occur with disruption of GM-CSF signaling at the level of GM-CSF production as in GM-CSF deficiency in mice, or the presence of neutralizing GM-CSF autoantibodies in patients with autoimmune PAP, the homozygous presence of genetic mutations in either GM-CSF receptor α or β chains that disrupt GM-CSF signaling. In secondary PAP, the syndrome is presumed to be caused by a reduction in either the numbers or functions of alveolar macrophages caused by any one of various underlying clinical disorders. In contrast, disorders of surfactant production caused by mutations in the genes encoding SP-B, SP-C, and ABCA3 (and likely others) result in the production of biochemically and functionally abnormal surfactant that disrupts alveolar structure resulting in gross parenchymal lung distortion. See text for details.

Figure 3. Relationship between GM-CSF autoantibody concentration, GM-CSF bioactivity, and GM-CSF dependent myeloid functions

At GM-CSF autoantibody concentrations below the critical threshold, GM-CSF bioactivity and GM-CSF dependent functions (i.e., CD11b stimulation, neutrophil phagocytosis) vary inversely with autoantibody concentration. At and above the critical threshold, GM-CSF bioactivity and GM-CSF-dependent functions are reduced minimum values (i.e., alveolar macrophage catabolism of surfactant). The hatched region represents the inverse relationship; the black bar represents priming caused by supranormal GM-CSF levels present during infection or exogenous administration; the white bar represents residual, non-zero minimal values of some function (e.g., phagocytosis). The critical threshold concept provides an explanation of why a high level of GM-CSF autoantibody is virtually diagnostic of autoimmune PAP and yet autoantibody levels (which are generally well above the critical threshold value) do not reflect lung disease severity. Adapted from reference [57].

Figure 4. Schematic of proposed mechanism by which GM-CSF regulates LPS responses in macrophages

GM-CSF initially binds to the low-affinity GM-CSF receptor α chain (α), followed by association with the affinity-enhancing β chain (β) and constitutively associated Janus kinase 2 (JAK 2) resulting in assembly of a dodecameric structure signaling complex. At low concentrations of GM-CSF (<300 pg/ml), as are normally present in normal healthy tissues, signaling occurs through Ser585 of the β chain and results in myeloid cell survival and GM-CSF stimulated expression of some GM-CSF-dependent TLR-4 signaling pathway components, CD14, RP105, and IRAK-M (dotted lines); other components are not affected by GM-CSF. Both GM-CSF-dependent and GM-CSF-independent components are required for normal responses to LPS. At high concentrations of GM-CSF (>300 pg/ml), signaling via Ser585 is extinguished and signaling occurs exclusively via residue Tyr577, activating STAT5 signaling, which enhances NFκB activity and the LPS response. Adapted from reference [15].

Figure 5. GM-CSF, via PU.1, uncouples microbial uptake from infection

A. Schematic of adenoviral infection pathway in normal epithelial cells and alveolar macrophages from GM-CSF deficient mice. Virions are internalized by receptor-mediated endocytosis and rapidly escape endosomal confinement via endosome-lysis, a mechanism dependent on both viral and host factors. Virions are then efficiently translocated to the nucleus where the nuclear injection of the viral genome results in viral replication and transduction of the cell. B. In alveolar macrophages from normal mice or from GM-CSF deficient mice after retroviral-transduction to restore PU.1 expression, adenoviral virions are rapidly internalized but cannot escape endosomal confinement, and undergo fusion with lysosomes forming phagolysosomes where virions are nearly completely destroyed without transduction of the cell. Ectopic expression of PU.1 in epithelial cells also blocks endosomal escape, and nuclear transduction and promotes viral clearance.