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. Author manuscript; available in PMC: 2014 Oct 10.
Published in final edited form as: N Engl J Med. 2014 Feb 19;370(10):911–920. doi: 10.1056/NEJMoa1307361

Figure 3. Effect of ADA2 Deficiency in Patients.

Figure 3

Cells stained with endothelial-cell activation marker E-selectin (green) are shown in a brain-biopsy sample from Patient 5 (Panel A) and in skin-biopsy samples from Patient 5 (Panel B) and a control donor (Panel C). E-selectin in endothelial cells (expressing von Willebrand factor [vWF; red]) indicates endothelial-cell activation (Panels A and B). E-selectin is absent in endothelial cells from a healthy control (Panel C). Nuclei are stained blue with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars indicate 20 µm. Interleukin-1β immunostaining (red) is shown in a brain-biopsy sample from Patient 5 (Panel D) and in skin-biopsy samples from Patient 4 (Panel E) and a control donor (Panel F). Positive cells can be seen in the brain sample from Patient 5 (Panel D), and robust interleukin-1β staining is seen in a skin-biopsy sample from Patient 4 (Panel E). An M1 and M2 differentiation assay (Panels G through J) was performed in monocytes that had been isolated by means of negative selection from blood samples from Patient 1 and an age-matched control. Equal cell numbers were seeded; M2 macrophage differentiation was induced with 50 ng per milliliter of macrophage colony-stimulating factor (M-CSF), and M1 macrophage differentiation with 20 ng per milliliter of granulocyte–macrophage colony-stimulating factor (GM-CSF) for 10 days. Control monocytes attached and differentiated, showing macrophage-like morphologic features under both M-CSF and GM-CSF stimulation (Panels G and H). Very few attached and differentiated M2-like cells were observed in M-CSF–stimulated monocytes from Patient 1 (Panel I). However, M1-like cells were observed in GM-CSF–induced differentiation of monocytes from Patient 1 (Panel J) — similar to those seen in control cells. Human dermal microvascular endothelial cells were grown to confluence and cocultured with monocytes isolated from a control donor and from Patient 1 for 3 days. Nonadherent cells were removed, and the endothelial-cell layers were stained for the endothelial junction protein VE-cadherin (red), F-actin (green), and DAPI (blue). Endothelial cells cultured with healthy control monocytes (Panel K) show normal cell layers, whereas endothelial cells cocultured with monocytes isolated from Patient 1 show damaged, interrupted endothelial cell layers (Panel L).