Figures
- Figure 1–
Radiographic and histopathological appearance of the lungs of the patients with pulmonary alveolar proteinosis caused by recessive CSF2RBS271L mutations. a) Posterior-anterior chest radiograph at the time of diagnosis, 9 yrs of age. Alveolar infiltrates are present throughout both lung fields. b) High-resolution computed tomography of the chest. Ground-glass opacification is superimposed on thickened interlobular and septal lines. c) Chest radiograph prior to an annual whole lung lavage therapy at 17 yrs of age. d) Bronchoalveolar lavage (BAL) fluid cytology at diagnosis (Papanicolaou stain). e) BAL fluid cytology at diagnosis (Periodic acid-Schiff (PAS) stain). f) Surgical lung biopsy obtained at diagnosis. Note the presence of PAS-staining material filling alveoli and also present in terminal airways. g) High-power view showing that alveolar wall architecture is normal (haematoxylin and eosin stain). d, e, g) Scale bars = 50 μm. f) Scale bar = 200 μm.
- Figure 2–
Nucleotide sequence of CSF2RB, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signalling, GM-CSF clearance and pedigree of the patient's family. a) PCR-based nucleotide sequencing of genomic DNA using CSF2RB-specific primers (5′-TCTCGGAGCTGTTGGACACA-3′, 5′-AAGATGCTCACCCTGCATCTG-3′). Amino acid sequence numbers are based on the human CSF2RB cDNA with first base of the ATG codon labelled +1 (Genbank accession number NM_000395). A single C>T substitution at nucleotide 812 results in a single amino acid substitution Ser>Leu at amino acid 271. The patient was homozygous and both parents were heterozygous for this mutation (CSF2RBS271L) while the brother did not carry the mutation. b) GM-CSF receptor signalling analysis. Fresh heparinised blood from the patient, a healthy individual (positive control) or a patient with recessive CSF2RAR217X mutations (negative control) was incubated alone (−) or with GM-CSF (GM), interleukin (IL)-2 or IL-3 (each at 10 ng·mL−1) for 15 min followed by Western blot analysis to detect phosphorylated signal transducer and activator of transcription (pSTAT5), total STAT5 or actin (to ensure equal loading of cell lysates). The IL-3 control demonstrates that the β-chain can function independently of the GM-CSF receptor α-chain. c) Evaluation of cell/receptor-mediated GM-CSF clearance. Blood leukocytes isolated from the patient (•) or a healthy individual (□) or dishes of culture media without cells (○) were incubated with GM-CSF (1 ng·mL−1) added at time 0. At subsequent times, GM-CSF concentration was measured in culture media by ELISA as described previously 4. #: significant differences in levels of GM-CSF in media in plates containing control cells compared to plates with no cells. d) Segregation of the CSF2RBS271L allele in the patient's family. •: homozygous individuals; ▓: heterozygous individuals; □: non-carriers of the CSF2RBS271L allele. The health status of each member is indicated and the arrow indicates the propositus.
- Figure 3–
Reproduction and function of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors harbouring the CSF2RBS271L mutation. Human embryonic kidney epithelial cells (293 cells) were evaluated alone (negative (-ve) control) or after transfection-mediated expression of a normal CSF2RA allele together with either a normal CSF2RB allele (positive (+ve) control), the patient's allele (CSF2RBS271L) or a previously proposed mutant CSF2RB allele (CSF2RBP603T; previously named P602T using a different naming convention 9). a) Cells were incubated with (+) or without (−) GM-CSF (10 ng·mL−1, 15 min) and evaluated by immunoprecipitation and Western blotting as described previously 4 to detect phosphorylated signal transducer and activator of transcription (pSTAT)5 or total STAT5. Note, the CSF2RBP603T -derived receptors resulted in signalling equal to the normal GM-CSF receptors. b) The same experiment was performed as in a) but increased GM-CSF concentrations were used for stimulation. Note, the partial signalling of CSF2RBS271L-deirved GM-CSF receptors. c) Evaluation of the effects of the CSF2RBS271L (•) or CSF2RBP603T (▾) alleles on cell-mediated GM-CSF clearance. GM-CSF (1 ng·mL−1) was added at time 0 and subsequently measured in culture media at the indicated times by ELISA. Note, the CSF2RBS271L-derived receptors cleared GM-CSF at an intermediate rate compared to healthy control (CSF2RBNormal; □) and that CSF2RBP603T-derived receptors had normal GM-CSF clearance. ○: no cells. d) Evaluation of the effects of CSF2RBS271L and CSF2RBP603T mutations on cell-mediated GM-CSF clearance. Results are similar to those in c) except that measurements were made 24 h after initiating GM-CSF exposure (n = 3 independent determinations in each group). p<0.01 for comparison to cell-free control. #: GM-CSF was undetectable.
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