To the Editors:
Pulmonary alveolar proteinosis (PAP) is a syndrome characterised by accumulation of surfactant in alveoli resulting in respiratory insufficiency 1. Surfactant homeostasis is critical for lung function and is tightly regulated, in part, by pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF), which is required for surfactant clearance by alveolar macrophages 2 and alveolar macrophage maturation 1. The effects of GM-CSF are mediated by cell-surface receptors composed of GM-CSF-binding α-chains and affinity-enhancing β-chains (encoded by CSF2RA and CSF2RB, respectively) 3. Ligand binding activates signalling via multiple pathways including the signal transducer and activator of phosphorylation (STAT)5 4. Disruption of GM-CSF signalling causes PAP by impairing surfactant catabolism in alveolar macrophages 1. In 90% of patients, PAP is caused by neutralising GM-CSF auto-antibodies 5, 6. Through the Rare Lung Diseases Network global PAP detection programme, we identified PAP caused by recessive CSF2RA mutations and developed novel diagnostic methods to identify patients with PAP caused by GM-CSF receptor dysfunction 4, 7. Herein, we report a case of hereditary PAP caused by disruption of GM-CSF receptor β-chain function.
A previously healthy 9-yr-old female presented with bilateral pneumonia, followed 3 months later by progressive dyspnoea of insidious onset. The diagnosis of PAP was suggested by chest radiograph findings, high-resolution computed tomography and bronchoalveolar cytology, and was confirmed by surgical lung biopsy. Pulmonary histopathology was typical of primary PAP (fig. 1) and she was successfully treated by serial whole lung lavage therapy. Details of the case history are included in the online supplement. A GM-CSF auto-antibody test was negative and the serum GM-CSF level was increased (25.9 pg·mL−1) suggesting GM-CSF receptor dysfunction as the molecular basis of PAP 4, 7. A molecular evaluation was undertaken and included GM-CSF receptor detection, STAT-5 phosphorylation, CSF2RA and CSF2RB nucleotide sequencing, and cloning as reported previously 4, 7. GM-CSF receptor α- and β-chains were detected on blood leukocytes from the patient and all family members by flow cytometry and Western blotting (data not shown) 4, 7. Nucleotide sequencing of leukocyte mRNA and genomic DNA revealed a normal CSF2RA sequence for the patient and all family members (data not shown). A single CSF2RB point mutation (c.812C>T) in exon 7 was identified in both mRNA and DNA from the patient (who was homozygous for the mutation) and from her parents (both heterozygous) but not from her brother (fig. 2a). This mutation caused a single amino acid change (p.Ser271Leu) in the GM-CSF receptor β-chain that impaired STAT5 phosphorylation in blood leukocytes following stimulation by GM-CSF and interleukin (IL)-3, but not IL-2 (fig. 2b). Since IL-3 stimulates STAT-5 phosphorylation via the β-chain common to GM-CSF, IL-3 and IL-5 receptors 8, we used IL-3 as an alternative means to demonstrate β-chain dysfunction in our patient (fig. 2b). Since IL-2 stimulates STAT5 phosphorylation independent of the β-chain, we used IL-2 as a positive control to demonstrate STAT5 phosphorylation in the patient's cells (fig. 2b). Consistent with disruption of GM-CSF receptor function, cell-mediated clearance of GM-CSF was impaired in the patient's leukocytes compared to those from a healthy control (fig. 2c). These results demonstrate that homozygous but not heterozygous CSF2RBS271L mutations impair GM-CSF receptor function in parallel with the occurrence of PAP (fig. 2d).
Gene cloning and expression of GM-CSF receptors in human 293 cells 4, 7 reproduced the signalling defect caused by the CSF2RBS271L mutation (fig. 3a). Interestingly, use of increased concentrations of GM-CSF for stimulation demonstrated partial functioning GM-CSF receptors derived from the CSF2RBS271L allele (fig. 3b). Consistent with this, cell-mediated clearance of GM-CSF by receptors harbouring this mutation was reduced but not absent (fig. 3c and d).
Previously, a CSF2RB-point mutation causing a substitution at amino acid 603 (P603T) was proposed as the molecular basis of PAP 10. We evaluated this mutation using the gene cloning approach described previously. The P603T mutation did not affect GM-CSF receptor function (fig. 3a) or cell-mediated GM-CSF clearance (fig. 3c and d). Furthermore, population studies showed it is present in ∼6% of the general population (NCPI single nucleotide polymorphism Database ID rs1801122; www.genecards.org). Based on these results, CSF2RBP603T represents a sequence polymorphism rather than a genetic cause of PAP as proposed 10. Thus, this is the first reported case of hereditary PAP due to CSF2RB mutations.
Our findings demonstrate that CSF2RB is critical for surfactant homeostasis in humans and that homozygous but not heterozygous CSF2RB mutations impairing GM-CSF receptor function cause a hereditary form of primary PAP. They extend prior findings that PAP is caused by the absence of GM-CSF receptor β in mice 10. The age at onset, presentation, biomarkers, pulmonary histopathology, natural history and response to whole lung lavage therapy are similar to those of hereditary PAP caused by CSF2RA mutations 4, 7. Except for the earlier age at onset and some biomarkers (increased serum GM-CSF and absence of GM-CSF auto-antibodies), many features are similar to those of patients with autoimmune PAP 1. The remarkable similarities in pathogenic, radiographic, histopathological and clinical findings in PAP caused by CSF2RA 4, 7, CSF2RB mutations or high levels of neutralising GM-CSF auto-antibodies in humans 1, by injection of patient-derived human GM-CSF auto-antibodies in healthy non-human primates 6 or by CSF2RB mutations or GM-CSF deficiency in mice 2, suggest the pathogenesis of each is similar and support the usefulness of grouping them together as primary PAP. The partial functioning of CSF2RBS271L-derived receptors suggests that aerosolised GM-CSF therapy may be of clinical benefit for this patient.
Footnotes
↵This manuscript has supplementary material available from www.erj.ersjournals.com
Support Statement
This work was supported, in part, by grants from the National Center for Research Resources (RR019498 to B.C. Trapnell in support of the Rare Lung Diseases Network), the National Heart, Lung and Blood Institute (HL085453 to B.C. Trapnell), the Division of Pulmonary Biology at the Cincinnati Children's Hospital (Cincinnati, OH, USA).
Statement of Interest
None declared.
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