Pulmonary alveolar proteinosis (PAP) is characterized by accumulation of surfactant in alveolar macrophages, resulting in respiratory insufficiency. Patients with PAP also present with abnormalities of myeloid cells leading to increased susceptibility to infections.
The surfactant is composed of lipids and proteins (surfactant proteins [SP]) and is produced by type II alveolar epithelial cells (AEC-II). Lipids (mostly phospholipids) make up 90 percent of surfactant and form bilayers that reduce surface tension at the air–liquid interface in the alveoli, thus avoiding alveolar collapse. Surfactant also contains hydrophobic proteins (SP-B, SP-C) that contribute to surfactant organization (Weaver and Conkright, 2001), as well as hydrophilic proteins (SP-A, SP-D) with antimicrobial properties (Kingma and Whitsett, 2006). Careful balance of surfactant production, reuptake, recycling, and catabolism by AEC-II and by alveolar macrophages maintains the surfactant pool at relatively stable levels.
Conditions with surfactant metabolism dysfunction in humans include disorders of surfactant production (DSP) and PAP. In the former, the composition of surfactant is affected because of mutations in genes that encode for SP-B, SP-C (Whitsett and Weaver, 2002) or for the ATP-binding cassette subfamily A member 3 (ABCA3) protein, a membrane lipid transporter (Shulenin et al., 2004). As a result of altered composition of surfactant and inability to lower alveolar surface tension, alveolar anatomy is distorted in patients with DSP, leading to respiratory failure. In contrast, surfactant composition is normal and alveolar architecture is largely preserved in patients with PAP, but surfactant accumulates in alveolar macrophages and in alveolar walls, leading to inflammatory changes (Trapnell et al., 2009). A series of observations in humans and mice have shown that defects of granulocyte macrophage-colony stimulating factor (GM-CSF)–mediated signaling play a critical role in the pathophysiology of PAP, regardless of the congenital, autoimmune, or secondary nature of the disorder.
Molecular Pathophysiology of PAP
The molecular pathophysiology of PAP was unraveled following the serendipitous discovery that disruption of the Csf2 gene (encoding for GM-CSF) in mice leads to a lung pathology that is similar to human PAP, with accumulation of foamy alveolar macrophages, periodic acid-Schiff (PAS)–positive material in alveolar walls, and peribronchial lymphocytic infiltrate (Dranoff et al., 1994; Stanley et al., 1994). While the material that accumulates in alveolar macrophages and walls was shown to be surfactant, and its composition to be normal, the accumulation of surfactant was found to be due to reduced clearance by alveolar macrophages; reuptake, recycling, and catabolism by AEC-II are not affected (Dranoff et al., 1994). Similar features were subsequently demonstrated in mice in which the csf2rb gene, encoding for the β chain of the GM-CSF receptor (GM-CSFR), had been disrupted (Robb et al., 1995).
Following this discovery, the effect of GM-CSF on the function of alveolar macrophage has been studied extensively. GM-CSF binds to a heterodimeric cell-surface receptor (GM-CSFR) that includes a GM-CSF–binding α chain and a signal-transducing common β chain (βc) shared by the receptors for interleukin (IL)-3 and IL-5 and known to bind the intracytoplasmic tyrosine kinase JAK2 (Hercus et al., 2009). Upon ligand binding, a dodecahedral complex is formed that consists of four molecules each of GM-CSFRα, βc, and JAK2 molecules (Hansen et al., 2008). At low GM-CSF concentrations, signaling through GM-CSFR results in myeloid cell survival and differentiation via activation of NF-κB and induction of bcl-2, whereas higher GM-CSF concentrations induce STAT5 phosphorylation and result in cellular activation and proliferation. This dual effect is mediated by βc phosphorylation at residues Ser585 or Tyr577, respectively (Guthridge et al., 2006). Importantly, the levels of GM-CSF in the lungs regulate expression of the transcription factor PU.1 in alveolar macrophages (Fig. 41.1) and promote adhesion, phagocytosis, expression of various surface receptors (Toll-like receptor [TLR]-2, TLR-4, Fc receptors, α- and β-integrins), secretion of inflammatory cytokines, microbial killing, and catabolism of surfactant (Shibata et al., 2001). These functions were defective in alveolar macrophages obtained from csf2-/- mice (Paine et al., 2001; Shibata et al., 2001) but could be rescued by retroviral-mediated expression of PU.1 (Shibata et al., 2001). Furthermore, the PAP pathology observed in csf2-/- mice can be rescued by restoring GM-CSF levels in the lungs, but not by its systemic administration (Huffman et al., 1996; Reed et al., 1999), indicating a critical role for pulmonary GM-CSF in alveolar macrophage function and surfactant homeostasis. Moreover, the lung phenotype of csf2rb-/- mice can be rescued by transplantation of bone marrow cells from wild-type donors (Nishinakamura et al., 1996) or of βc-deficient bone marrow cells transduced with a βc-expressing retroviral vector (Kleff et al., 2008), indicating the critical role of lung macrophages (of hematopoietic origin) rather than AEC-II cells in the pathophysiology of the disease.
GM-CSF–mediated signaling brings together innate and adaptive immune responses. It induces secretion of IL-18 and IL-12 by macrophages. These cytokines stimulate Th1 and NK lymphocytes to produce IFN-γ, thus prompting immune responses against intracellular pathogens (Berclaz et al., 2002). In keeping with this, csf2-/- mice are highly susceptible (and show increased mortality) to a variety of pathogens, including Streptococcus, Pseudomonas, Listeria, Pneumocystis jiroveci, M. tuberculosis, and adenovirus (Ballinger et al., 2006; Carey et al., 2007; Gonzalez-Juarrero et al., 2005; LeVine et al., 1999; Shibata et al., 2001). These infections are not strictly confined to the lungs, indicating that impaired GM-CSF–mediated signaling results in systemic defects of immune responses. Altogether, these data from csf2-/- mice clearly indicated that GM-CSF plays a critical role in the regulation of alveolar macrophage differentiation and function and in immune defense. Furthermore, the striking similarities in lung pathology between csf2-/- mice and patients with PAP suggested that congenital or acquired abnormalities of GM-CSF–mediated signaling play a role in the pathophysiology of the disease in humans.
PAP: Classification and Molecular Features
The first case of PAP in humans was reported in 1958 (Rosen et al., 1958), but its pathogenesis remained obscure for several decades until csf2-/- mice were generated and characterized. Current classification of human PAP includes autoimmune, congenital, and secondary forms (Trapnell et al., 2009).
In 1999, Kitamura et al. reported neutralizing anti–GM-CSF autoantibodies in the serum of patients with PAP (Kitamura et al., 1999). Autoimmune PAP accounts for 90 percent of all cases of PAP (Inoue et al., 2008), yet its prevalence is low (3 or 4 individuals per million). Usually, clinical manifestations begin in the third or fourth decade of life, and lung pathology is characterized by accumulation of PAS-positive material in the alveoli that maintain a normal architecture. Patients with autoimmune PAP are also at high risk of infections, which account for 18 percent of the deaths (Seymour and Presneill, 2002). The antibodies detected in patients with autoimmune PAP bind GM-CSF with high affinity (Uchida et al., 2004). Interestingly, it has recently been shown that GM-CSF autoantibodies are present also in healthy donors (Uchida et al., 2009) and in pharmaceutical immunoglobulin preparations (Svenson et al., 1998). However, the levels of GM-CSF autoantibodies are much higher in patients with autoimmune PAP, leading to the hypothesis that they may play a pathogenic role when their concentration exceeds a certain threshold (Bendtzen et al., 2007), estimated to be more than 10.4 μg/m: (Uchida et al., 2009). Injection of GM-CSF autoantibodies isolated from patients with autoimmune PAP into nonhuman primates can in fact reproduce the features of the disease if autoantibody levels are maintained above 40 μg/mL for several months (Sakagami et al., 2009).
Following the demonstration that disruption of the csf2 and csf2rb genes in mice leads to a PAP phenotype, defects in the same genes were sought in humans with PAP. In 1997, four infants with an established or putative diagnosis of PAP were reported in whom lack of βc expression was demonstrated on the surface of peripheral leukocytes (Dirksen et al., 1997). Expression of the GM-CSFRα chain was preserved, but binding of GM-CSF and in vitro progenitor clonogenic assays showed altered response to GM-CSF. Homozygosity for a single amino acid substitution (Pro603Thr) was identified in one of the three patients studied, but this was then found to represent a polymorphism (Freeburn et al., 1998). No defects in the CSF2RB gene were identified in the remaining patients, leaving the molecular basis of the disease unclear. However, more recently, a homozygous missense mutation in the CSF2RB gene, resulting in S271L amino acid substitution, was identified in a female child presenting with pneumonia and progressive dyspnea. The mutation prevented STAT5 phosphorylation in blood leukocytes following stimulation by GM-CSF and IL-3 (Suzuki et al., 2011). While this observation extends to humans previous observations in mice that csf2rb mutations may lead to a PAP phenotype, this remains a very rare cause of the disease.
In contrast, two groups have provided conclusive evidence that PAP may be caused by genetic defects of the CSF2RA gene, which is located in the pseudoautosomal region of chromosome X, at Xp22.3, and at the tip of the short arm of chromosome Y, at Yp11.32. Susuki et al. (2008) described two sisters aged 6 and 8 years with growth failure, tachypnea, and severe pulmonary restrictive impairment. Chest radiograph and histopathological examination of a lung biopsy were consistent with the diagnosis of PAP. Search for GM-CSF autoantibodies was negative. The concentrations of GM-CSF in the bronchoalveolar lavage (BAL) fluid, and of SP-D in the serum, were markedly elevated. Although expression of both GM-CSFRα and βc chains at the surface of leukocytes was normal, GM-CSF–induced upregulation of CD11b was impaired and expression of a low-molecular-weight form of GM-CSFRα chain was demonstrated by Western blotting. Genetic analysis revealed that both sisters carried a 1.6 Mb deletion in the pseudoautosomal region 1 of the maternally derived X chromosome and a single nucleotide mutation, leading to Gly174Arg substitution, in the paternally derived CSF2RA gene. When co-transfected with βc into 293 cells, the GM-CSFRα chain mutant failed to internalize GM-CSF added to the culture and resulted in markedly decreased phosphorylation of STAT5 in response to GM-CSF. This defect was partially rescued at higher GM-CSF concentrations (Suzuki et al., 2008). Martinez-Moczygemba et al. described a 4-year-old girl with Turner syndrome and respiratory insufficiency; pathological findings at lung biopsy and on BAL were also consistent with the diagnosis of PAP. Search for GM-CSF autoantibodies was negative, but serum GM-CSF concentration was increased more than 200-fold. A complete lack of GM-CSFRα chain expression on the surface of peripheral blood monocytes was demonstrated, and expression of CD11b was not upregulated in response to GM-CSF. Molecular cytogenetic analysis showed a 46Xi(Xq) karyotype, with truncation of the Xp arm and lack of pseudoautosomal region 1 on the i(Xq) chromosome. The observation that the GM-CSFRα chain transcript could not be demonstrated by RT-PCR prompted a search for mutation on the other allele of the CSF2RA gene; a deletion of the genomic segment encompassing exons 5–13 was demonstrated (Martinez-Moczygemba et al., 2008). Altogether, these observations provide the first evidence that biallelic mutations of the CSF2RA gene cause congenital PAP in humans. Since then, seven additional children, all girls, have been identified with congenital PAP due to CSF2RA mutations (Trapnell et al., 2009). Elevated GM-CSF levels were consistently demonstrated in every patient.
Finally, PAP may also occur in association with various hematological disorders (myelodysplastic syndromes, leukemia, lymphoma, etc.), immunological diseases (severe combined immunodeficiency, IgA deficiency), infections (cytomegalovirus, M. tuberculosis, P. jiroveci, others), lysinuric protein intolerance, hematopoietic cell transplantation, and exposure to toxic substances and elements (Chung et al., 2009; Seymour and Presneill, 2002; Trapnell, 2003). The pathogenesis of secondary PAP remains poorly defined and may reflect acquired depletion or functional defects of alveolar macrophages.
Clinical Features, Diagnosis, and Treatment
The main clinical features of PAP include progressive respiratory failure with hypoxemia and growth arrest. Chest radiographs and computed tomography show diffuse and patchy opacities (Ishii et al., 2009; Trapnell et al., 2003). Open lung biopsies demonstrate accumulation of granular PAS-positive proteinaceous material in the alveoli and in the alveolar walls, foamy macrophages, and inflammatory infiltrates, without disruption of the lung architecture. Foamy macrophages and PAS-positive material are also recovered following BAL. In addition, patients with PAP suffer from increased susceptibility to pulmonary and extrapulmonary infections. These include both community- and hospital-acquired microorganisms (S. pneumoniae, H. influenzae, Klebsiella, Pseudomonas) and opportunistic pathogens (Nocardia, Mycobacteria). In a review of cases reported in the literature between 1958 and 1997, infections accounted for 18 percent of deaths (Seymour and Presneill, 2002).
Some clinical and laboratory features distinguish congenital PAP from autoimmune and secondary forms. The seven patients with molecularly proven CSF2RA mutations were 2 to 11 years old at the time of diagnosis (Trapnell et al., 2009); in contrast, autoimmune PAP typically affects subjects aged 30 to 40 years. Furthermore, no underlying disorders can be identified in patients with congenital PAP. At variance with autoimmune PAP, patients with congenital PAP do not have GM-CSF autoantibodies, and their GM-CSF serum levels are significantly elevated. In addition, levels of SP-D in the serum and of inflammatory chemokines and cytokines (MCP-1, GM-CSF, M-CSF) in BAL fluid are increased in patients with PAP, regardless of the congenital, autoimmune, or secondary nature of the disease (Trapnell et al., 2009).
Whole-lung lavage (i.e., large-volume BAL) is often used as the first line of treatment in patients with PAP and results in significant clinical and radiographic improvement (Michaud et al., 2009). However, approximately 15 percent of the patients (including those with congenital PAP) have a relapsing course and may require multiple rounds of the procedure. The observation that some patients with congenital PAP have residual GM-CSF–mediated signaling suggests that aerosolized delivery of GM-CSF might be therapeutic in these patients (Suzuki et al., 2008). Finally, because of the critical role of alveolar macrophage dysfunction in the pathogenesis of congenital PAP, hematopoietic stem cell transplantation (HSCT) and CSF2RA gene transfer might be considered. Indeed, one of the patients with congenital PAP due to CSF2RA mutations received HSCT from a matched unrelated donor but died from a severe respiratory infection shortly after transplantation, before immune reconstitution was achieved (Martinez-Moczygemba et al., 2008).
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