Impaired formation of neutrophil extracellular traps in patients with MDS

Abstract

Neutrophil extracellular traps (NETs) are networks of extracellular fibers primarily composed of DNA and histone proteins, which bind pathogens. We investigated NET formation in 12 patients with myelodysplastic syndrome (MDS) and 15 age-adjusted normal controls after stimulation with phorbol-12-myristate-13-acetate (PMA). Histones and neutrophil elastase were visualized by immunostaining. Since NET formation is triggered by reactive oxygen species (ROS), mainly produced by reduced NADP-oxidase and myeloperoxidase (MPO), ROS were analyzed by flow cytometry using hydroethidine, 3’-(p-ami-nophenyl) fluorescein, and 3’-(hydroxyphenyl) fluorescein. On fluorescence microscopy, PMA-stimulated MDS neutrophils generated fewer NETs than controls (stimulated increase from 17% to 67% vs 17% to 85%) (P 5 .02) and showed less cellular swelling (P 5 .04). The decrease in mean fluorescence intensity (MFI) of 4’,6-diamidino-2-phenylin-dole, indicating chromatin decondensation, was significantly less in MDS neutrophils than controls (DMFI 3467 vs DMFI 4687, P 5 .03). In addition, the decrease in MFI for fluorescein isothiocyanate, indicating release of neutrophil elastase from cytoplasmic granules, was diminished in patients with MDS (P 5 .00002). On flow cytometry, less cell swelling after PMA (P 5 .02) and a smaller decrease in granularity after H2O2 stimulation (P 5 .002) were confirmed. PMA-stimulated ROS production and oxidative burst activity did not reveal significant differences between MDS and controls. However, inhibition of MPO activity was more easily achieved in patients with MDS (P 5 .01), corroborating the notion of a partial MPO defect. We conclude that NET formation is significantly impaired in MDS neutrophils. Although we found abnormalities of MPO-dependent generation of hypochloride, impaired ROS production may not be the only cause of deficient NETosis in MDS.