Pulmonary alveolar proteinosis in adenosine deaminase–deficient mice

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Pulmonary alveolar proteinosis in adenosine deaminase–deficient mice Rupreet Dhanju, MSc, Weixian Min, MSc, Cameron Ackerley, PhD, Lorand Cimpean, DVM, Nades Palaniyar, MSc, PhD, Chaim M. Roifman, MD, FRCPC, FCACB, Eyal Grunebaum, MD Journal of Allergy and Clinical Immunology Volume 133, Issue 5, Pages 1467-1471.e4 (May 2014) DOI: 10.1016/j.jaci.2013.11.029 Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Surfactant accumulation in the alveoli of 16- to 18-day-old ADA-KO mice, which is prevented by PEG-ADA treatment. A, Hematoxylin and eosin demonstrates a granular eosinophilic substance (arrows) in the alveoli of ADA-KO mice (middle), not present in littermate control mice (left) or ADA-KO mice treated with PEG-ADA (right); ×20 magnification. B, Periodic acid–Schiff (PAS)-stained substance (left) in the lungs of ADA-KO mice is diastase resistant (right); ×40 magnification. Enlargements are ×100 magnification. Bar = 200 μm. C, Electron microscopy demonstrates accumulation of a granular substance and AMs in the alveoli of ADA-KO mice (×3000 magnification, bar = 10 μm). Figures representative from N = 5 mice of each genotype. Journal of Allergy and Clinical Immunology 2014 133, 1467-1471.e4DOI: (10.1016/j.jaci.2013.11.029) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Increased apoptosis of AMs from ADA-KO mice. Representative flow cytometry analysis (left) and bar graph (right, mean ± SD) of AMs from 16- to 18-day-old ADA-KO and littermate control mice assessed for Annexin V (A) and propidium iodide (B) (PI) intercalation. Results are representative of 3 independent experiments. C, Electron microscopy analysis of AMs from ADA-KO mice demonstrating nuclear chromatin fragmentation and bleb formation typical of apoptosis. Figures are representative of 40 images; ×15,000 magnification. Bar = 3 μm. Journal of Allergy and Clinical Immunology 2014 133, 1467-1471.e4DOI: (10.1016/j.jaci.2013.11.029) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Alveolar macrophages from ADA-KO mice are foamy and contain inclusion bodies, lamellar structures, and cholesterol crystals. Electron microscopy of AMs from 16- to 18-day-old ADA-KO mice. A, AMs are foamy and filled by inclusion bodies (×5,000 magnification, bar = 10 μm). B, AMs from ADA-KO mice contain many concentric “lamellar-like” structures (×10,000 magnification, bar = 5 μm). C, Cholesterol crystals (×20,000 magnification, bar = 1 μm). Figures are representative of 40 independent images (N = 5 mice). Journal of Allergy and Clinical Immunology 2014 133, 1467-1471.e4DOI: (10.1016/j.jaci.2013.11.029) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Increased Oil-Red-O (ORO) staining of AMs in the lungs of ADA-KO mice. A, Excessive ORO staining of AMs from ADA-KO mice compared with littermate control mice. Figures representative from N = 5 mice of each genotype (×10 magnification, bar = 50 μm). B, Percentage of ORO-stained cells among AMs from ADA-KO mice, ADA-KO mice treated with PEG-ADA, and littermate control mice. Graph bar represents mean ± SD of 500 cells for each group from 4 independent experiments. Journal of Allergy and Clinical Immunology 2014 133, 1467-1471.e4DOI: (10.1016/j.jaci.2013.11.029) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 AMs of ADA-KO mice demonstrate normal phagocytosis. Uptake of IgG-coated beads by AMs from 16- to 18-day-old ADA-KO mice (left) is similar to that from littermate control mice (right). Images in the top row are differential interference contrast (DIC), the second row are Cy3-conjugated anti-IgG antibodies, and the third row are stained with 4′-6-diamidino-2-phenylindole, dihydrochloride (DACI). The bottom row shows merged images. Blue arrows denote intracellular beads, and red arrows indicate extracellular beads. Figures are representative of more than 35 images from 3 independent experiments for each genotype (×40 magnification, bar = 40 μm). Journal of Allergy and Clinical Immunology 2014 133, 1467-1471.e4DOI: (10.1016/j.jaci.2013.11.029) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions