22  Pulmonary Alveolar Proteinosis Syndrome	391

of these pathognomonic GM-CSF targeting antibodies led to a nomenclature change from “Idiopathic” PAP to the more appropriately named autoimmune PAP [1, 18–21]. These studies identi ed the critical role that GM-CSF plays in maintaining surfactant homeostasis, alveolar stability, and host defense [22].

Epidemiology

While disorders of surfactant homeostasis are rare, they occur in worldwide distribution. More than 1000 cases of PAP have been reported in the medical literature since it wasrst reported in 1958 [2, 4, 5, 9, 23–26]. Autoimmune PAP accounts for 90% of all patients with PAP [2, 4–6, 23]. A national PAP registry study that identi ed 248 patients with PAP syndrome in Japan reported the incidence and prevalence of autoimmune PAP to be 0.49 ± 0.13 and 6.7 per million, respectively [23]. Another study conducted the United States based on analysis of comprehensive health insurance claims data reported the prevalence of PAP syndrome to be 6.87 per million [27]. Interestingly, 31% of the patients in the Japanese national registry were identi ed through mandatory health screening and were asymptomatic at diagnosis suggesting these prevalence values may be underestimates. Indeed, a third more recent study from Japan based on Poisson analysis of annual incidence data reported a prevalence of 26.

Male gender and smoking have been described as risk factors for PAP [2]. However, an analysis of several large case studies found that 21–43% of PAP patients were never smokers, indicating that PAP is not simply a smoking-related disease [2, 4–6, 23]. Furthermore, while a comprehensive meta-analysis of 410 separate cases of PAP found that patients were more likely to be male (male: female ratio = 2.65:1.0), the gender balance was reversed when non-­ smoking patients were taken in isolation (male: female ratio = 0.69:1.0). This led to the conclusion that the male dominance among patients with PAP may be explained by their higher frequency of tobacco use. The median age at diagnosis in this study was 39 years (39 in men and 35 in women) [2]. Finally, several studies have searched for a genetic predisposition to the development of autoimmune PAP [28, 29]; one evaluating 198 autoimmune PAP patients and 395 controls of Japanese ancestry identi ed two major histocompatibility complex alleles as independent risk factors (HLA-DRB1*08:03 and HLA-DPb1) [17].

In summary, PAP syndrome is caused by autoimmune PAP in 90% of cases and has a prevalence of 7–10 (possibly up to 26) per million in the general population. It affects

men, women, and children independent of ethnicity, gender, and socioeconomic status [22].

Pathogenesis

Surfactant Homeostasis in PAP

A primary function of surfactant is to reduce surface tension at the air–liquid–alveolar wall interface, thereby preventing alveolar collapse during the breath cycle. It is composed of ~80% polar phospholipids, ~10% neutral lipids (mostly cholesterol), and ~10% surfactant proteins. Surfactant is synthesized in type II alveolar epithelial cells (AEC-2) and secreted into the alveolar space. Surfactant is cleared from the lung surface by recycling and catabolism in AEC-2 cells and by catabolism and export by alveolar macrophages [7]. Studies in animals and humans identi ed a critical role for GM-CSF in surfactant homeostasis; alveolar macrophages require GM-CSF in order to clear surfactant (Fig. 22.2). GM-CSF is a 23-kDa cytokine identi ed in the 1970s [30, 31], which is expressed by a variety of cell types, including lung epithelial cells [32, 33]. It is, perhaps, best known its role in infammation, host defense, and autoimmune diseases [34, 35]. Speci cally, pulmonary GM-CSF stimulates survival, proliferation, differentiation, and the functions of alveolar macrophages [36–39].

GM-CSF Signaling Disruption

GM-CSF signaling occurs via cell surface receptors composed of a low af nity GM-CSF-binding α chain (CDw116) and an af nity-converting β chain (CD131) [40, 41] which constitutively binds Janus activating kinase (JAK) [42]. Ligand binding results in the assembly of αβJAK2 multimers and activation of JAK2, phosphorylation of α [43] and β chains, and initiation of signaling via multiple pathways, [42, 44] including the signal transducer of activation and transcription-5 (STAT5) [45].

Disruption of GM-CSF signaling causes the development of PAP (Fig. 22.3). Mice de cient in GM-CSF (Csf2KO) or the GMC-CSF receptor α chain (Csf2raKO) or β chain (Csf2rbKO) develop PAP with similar physiological, histopathological, and immunological features to autoimmune PAP [14, 15, 46]. PAP was corrected by transplantation of wild-type bone marrow in Csf2rbKO mice identifying myeloid cells and not lung epithelial cells as the cellular site of pathogenesis [47]. Reversal of PAP was achieved in Csf2KO mice by expression of GM-CSF in the lungs [48–50] and in

392								M. O’Callaghan et al.
		Phospholipids (PL)		SP-B		SP-C	Cholesterol (C)	Air
PL, C,						Alveolar		Liquid
	Tubular myelin					macrophage
SP-B,				Surfactant-containing
SP-C								Phospholipid
		Small surfactant		phagocytic vesicles
	SP-A							catabolism
		aggregates
Secretion	SP-D
			Lipid		Cholesterol		Cholesterol
			vesicle
				sequestration			efflux
			Esterified
ABCA3						ACAT
	Phagocytic		cholesterol	EC	EC			ABCG1
						Cholesterol
	vesicle		GM-CSF		ADRP	NCEH		ABCA1
	SP-A		receptor	JAK2				SRB1
SP-B	SP-D				PU.1	PPARγ		Apo-E
SP-C		Lysosome					OH-C
						LXR		27-OHase

Cholesterol	Type II	Type I	Capillary	Basement	Cholesterol
			endothelial cell	membrane
Liver	Alveolar epithelial cells				Liver
Congenital PAP			Primary PAP	Secondary PAP
(mutations in SFTPB, SFTPC,		(GM-CSF autoantibodies,		(Diseases/conditions that
ABCA3, TTF1, others)		mutations in CSF2RA/B)		reduce macrophage numbers or functions)	Blood

Fig. 22.2  Normal alveolar surfactant homeostasis. A portion of a pulmonary alveolus is shown. Pulmonary surfactant normally comprises a thin layer of polar lipids (mostly phospholipids), neutral lipids (mostly cholesterol), and surfactant proteins (A–D) located at the alveolar air– liquid interface that reduces surface tension on the alveolar wall and is critical to alveolar stability and lung function. Surfactant homeostasis is

tightly regulated by balanced secretion of surfactant lipids and proteins by type II alveolar epithelial cells and removal by these cells (through recycling and catabolism) and by alveolar macrophages (through catabolism and excretion). Alveolar macrophages require GM-CSF constitutively to an adequate rate of cholesterol clearance, which occurs through multiple pathways

Csf2rbKO and Csf2raKO mice by pulmonary macrophage transplantation [51, 52]. GM-CSF is detectable at low but non-zero levels in normal human lung [37]. While the absence of GM-CSF causes PAP, excessive GM-CSF causes accumulation of alveolar macrophages within the distal airspaces of the lung; a phenotype resembling desquamative interstitial pneumonia in humans [53]. In summary, pulmonary GM-CSF is a low abundance cytokine regulator of surfactant homeostasis, alveolar structure, lung function, and host defense [22].

Myeloid Cell Dysfunction

We have established that pulmonary GM-CSF plays a critical role in surfactant homeostasis, alveolar structure, and lung function via its regulation of myeloid cells—particularly alveolar macrophages [22]. Constitutive GM-CSF signaling occurs via PU.1, the master macrophage transcription factor

purine box binding protein 1 [37], and peroxisome proliferator-­activated receptor gamma (PPARγ) [54, 55], the lipid metabolism-related transcription factor. The transcription factor PU.1 is a required component of the GM-CSF signaling pathway, [37] and both PU.1 and PPARγ are needed for alveolar macrophage speci cation and to enable cholesterol exportation, surfactant clearance, and other important macrophage functions [30]. Furthermore, macrophages from Csf2KO mice have reduced expression of PPARγ and its target gene ATP binding cassette transporter G1 (ABCG1), a transmembrane lipid transporter protein important in cholesterol effux from macrophages [56–58]. And mice de cient in ABCG1 (ABCG1KO) develop a PAP-like pulmonary phenotype with foamy macrophage formation, implicating ABCG1 speci cally in pulmonary surfactant homeostasis [59, 60].

Similar observations have been made in human studies. Alveolar macrophages from patients with PAP are foamy in appearance due to reduced expression of PU.1, PPARγ, and ABCG1, associated with accumulation of esteri ed choles-

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22  Pulmonary Alveolar Proteinosis Syndrome

393

Air

Alveolar

Liquid

macrophage

Surfactant

EC

EC

Phospholipid

EC

containing

EC

vesicles

EC

catabolism

EC

EC

EC

EC EC

EC

EC

EC EC EC EC

EC

EC

Cholesterol

EC

EC

EC

Cholesterol

EC

sequestration

EC

efflux

Lipid

EC EC

ACAT

EC

ABCG1

EC

vesicles

EC

Cholesterol

ABCA1

EC

ADRPECNCEH

EC

EC

SP-C

EC

EC

SRB1

SP-B

SP-D

PU.1

PPARγ

EC

EC

Apo-E

SP-C

LXR

OH-C

27-OHase

Cholesterol

Type II alveolar

Type I alveolar

Capillary

Basement Cholesterol

epithelial cell

epithelial cell

endothelial cell

membrane

Liver

GM-CSF

Liver

autoantibodies

Blood

Fig. 22.3  Pathogenesis of autoimmune PAP. An increase in polyclonal, neutralizing GM-CSF autoantibodies blocks GM-CSF signaling in the blood and tissues, which impairs multiple functions by altering expression of multiple genes including expression of the macrophage cholesterol transporter, ABCG1, resulting in a primary reduction in cholesterol clearance and a secondary reduction surfactant clearance from the

alveolar surface. Increased intracellular cholesterol stimulates increases in ABCA1 and other LXR-mediated cholesterol clearance pathways that attempt albeit unsuccessfully to compensate for the loss of ABCG1mediated clearance. In response to reduced cholesterol effux, alveolar macrophages esterify and store cholesterol in lipid droplets, which accumulate inside the cells resulting in the formation of foam cells

terol in intracytoplasmic droplets [55, 58]. While surfactant from PAP patients contains increased amounts of phospholipids, the increase in cholesterol is even greater, resulting in an increase in the ratio of cholesterol to phospholipid [30, 31]. This has important implications for the surface tension-­ lowering biophysical properties of surfactant [61–63]. GM-CSF via its downstream pathways involving PU.1, PPARγ, and ABCG1 in alveolar macrophages regulates cholesterol effux as well as fatty acid catabolism but has no apparent effect on the initial steps involved in catabolism of phospholipids. In both human and murine studies this results in cholesterol esterlled intracytoplasmic droplets accumulating with alveolar macrophages or the so-called foamy macrophages [64]. Finally, this is further supported by the increase in alveolar macrophage surfactant cholesterol: phospholipid ratio when there is a loss of GM-CSF signaling, which impairs surfactant function [7].

Neutrophils from patients with autoimmune PAP and Csf2KO mice also have impaired host defense functions but

appear to be fully differentiated with normal morphology and phenotypic marker expression but reduced capacity for bacterial killing, phagocytosis, reactive oxidase species production, and cell adhesion [18]. Ex vivo exposure of normal human neutrophils to GM-CSF autoantibodies reproduces the functional impairments observed in PAP neutrophils [18]. It is likely that GM-CSF constitutively regulates the basal functional capacity of neutrophils in vivo in a rheostatic manner based on studies in humans, mice, and non-­ human primates [18]. The dysfunctional neutrophils in autoimmune PAP contribute to impaired innate immunity, thus providing a molecular and cellular explanation the increased infection risk in these patients [18].

GM-CSF Autoantibodies

Following the discovery in Csf2KO mice, elevated levels of GM-CSF autoantibodies were noted in the serum and BAL