CGD neutrophils have impaired NET formation by pyocyanin.
A) Comparison of pyocyanin-induced NET formation among three X-CGD patients, one p47-deficient CGD patient and their normal controls. Adherent non-CGD or CGD neutrophils (patients 1,3,4, 5) were exposed to 0–30 µM pyocyanin, 100 nM PMA or 0.1 U/mL glucose oxidase (GO), Sytox Orange fluorescence was monitored for 3.5 hrs and NET formation was quantified. The numbers in the boxes show residual superoxide productions of CGD neutrophils as % of the average of normal donors (100%). B) Images of adherent neutrophils of CGD patient 3 and a non-CGD control (Sytox Orange fluorescence) after 3.5 hour-exposure to 10, 30 µM pyocyanin, 0.1 U/mL glucose oxidase (GO) or 100 nM PMA. C) Images of NETs formed by p47-deficient CGD neutrophils and normal controls exposed to the same stimuli as previously. Magnified micrograph in the upper right corner shows normal neutrophils in different stages of NET formation at the end (3 hrs) of the Sytox Orange microplate assay. Examples for different stages are marked by white arrowheads and numbers. 1– These cells did not produce NETs. At 3 hrs their plasma membrane became somewhat permeable and Sytox Orange stains their resting multilobular nucleus. 2- These cells are in an intermediate phase. The plasma membrane is permeable, the typical lobulated nuclear morphology is lost but the integrity of the cell is still observed. 3– PMNs in the final stage of NETosis. The nuclear and cell morphology is completely lost, the DNA was released around the cell and it covers a larger area than the original cell size. Due to the weak fluorescent signal of NETs a longer exposure time had to be chosen and PMNs in stages 1 and 2 are overexposed. PMA, phorbol-myristate-acetate; PMN, polymorphonuclear neutrophil.