PMC5884887_01
Characteristic aberrations in neutrophil granulocytes from patients with specific granule deficiency. (A) Forward/sideward scatter of whole blood after erythrocyte lysis. (B) Hematoxylin/eosin staining of peripheral blood smear (left) and bone marrow aspirate (right) of the patient. (C) Immunofluorescence analysis of myeloperoxidase (MPO, red) and lactoferrin (LTF, green) in the neutrophils of patient (left) and healthy donor (right), scale bar: 5 microm. (D) Analysis of patient (left) and healthy control (right) neutrophils in transmission electron microscopy; scale bar: 2 mum.
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F1
Flow cytometric analysis of peripheral blood and bone marrow revealed a severe reduction of neutrophil granularity, which results in clustering of neutrophils with monocytes in the forward sideward scatter (Figure 1A). Morphologically, neutrophils presented hyposegmented with bilobed nuclei (Figure 1B, left panel) which was confirmed in bone marrow aspiration (Figure 1B, right panel). The protein lactoferrin, which is normally present in secondary granules of neutrophils, was undetectable in patients' neutrophils (Figure 1C). Electron microscopic analysis revealed a reduction in the number of granules in patients' neutrophils in comparison with HCs (Figure 1D).
Granule distribution, protein content, and glycosylation pattern of neutrophil granules are modified in specific granule deficiency (SGD). (A) Immunoelectron microscopy shows presence of lactoferrin (15 nm gold particles) in secondary granules in healthy controls (HCs, upper panel) and its absence in two SGD patients (two lower panels). Elongated sacs as well as aggregates of granules are observed in SGD but not in HCs (see zoomed in panels). Scale bars: 500 nm. (B) MPO-containing granules (red) are aggregated and polarized in SGD compared with HCs as shown by confocal immunofluorescence. Scale bar: 10 microm. (C) Lysozyme (green) co-localizes with MPO (red) in neutrophils of SGD patients (lower panel) but not of HCs (upper panel). (D,E) Different populations of granules are recognized specifically by the lectins (green) peanut agglutinin (PNA) (D) and UEA-I (E) in HCs (upper panel), displaying, respectively, no or partial co-localization with MPO (red). The granules in SGD neutrophils (lower panel) are not recognized by PNA (D) and react weakly with Ulex europaeus agglutinin I (UEA-I) (E) compared with HCs. (F) The anti-CD15 (green) staining is reduced in neutrophils of SGD patients (right) compared with healthy donor (left). No staining is visible in a lymphocyte in the healthy donor (asterisk). Scale bars (C-F): 5 microm.
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F3
Neutrophil chemotaxis and phagocytosis were unaltered in the patients (Figures S3C,D in Supplementary Material), further no difference in apoptosis could be detected (data not shown).