Figure 2. Functional studies on PLS neutrophils.

(A) Flow cytometry of nonstimulated and fMLF-stimulated (10^–8 M) neutrophils from PLS and a normal control demonstrating equal disappearance of CD62L and upregulation of CD11b in response to stimulation. Respiratory burst was quantified as DHR-positive cells by flow cytometry of isolated neutrophils stimulated by fMLF (10^–8 M). (B) NETosis. Neutrophils were stimulated with PMA or glucose oxidase (GO) to undergo NETosis and stained with DAPI and antibodies against MPO. NETosis was quantified as an increase in MPO-positive areas normally caused by formation of NETs with bound MPO. While some PLS neutrophils had increased MPO-positive areas after stimulation with glucose oxidase, these cells did not form NETs. The increased MPO-positive areas in these cells were caused by the presence of MPO in decondensated nuclei, as seen in the panels with ×100 magnification. Error bars for the PLS neutrophils demonstrate SD between the 2 experiments performed. One representative experiment out of 3 is shown for quantification of NETosis from the normal controls, and here error bars indicate SD between the 20 images used for quantification. (C) Processing of hCAP-18 by ionomycin-stimulated neutrophils as described in Methods. Cells and medium from ionomycin-stimulated (Io) or unstimulated neutrophils (Us) were blotted with a rabbit antibody against hCAP-18 (34). These functional studies (except the respiratory burst experiment, which was performed only once) were performed on 2 independent occasions 3 months apart with similar results.