Inhibition of spontaneous neutrophil apoptosis by parabutoporin acts independently of NADPH oxidase inhibition but by lipid raft-dependent stimulation of Akt

Abstract

Neutrophil cell death plays a crucial role in neutrophil homeostasis and the resolution of inflammation. The superoxide-producing NADPH oxidase is involved in pathogen degradation and subsequent activation of cell death programs. Neutrophils from patients with chronic granulomatous disease, who have a deficient NADPH oxidase activity, have been demonstrated previously to have a prolonged lifespan, suggesting that a basal NADPH oxidase activity also regulates spontaneous neutrophil turnover. The NADPH oxidase inhibitor parabutoporin (PP) does delay spontaneous apoptosis, but this effect is completely independent of NADPH oxidase inhibition. Instead, the prosurvival effect of PP depends on activation of protein kinase B/Akt via lipid raft signaling. Disruption of lipid rafts abrogates the prosurvival effect without interfering with NADPH oxidase activity. Furthermore, we cannot detect a different rate of spontaneous apoptosis between normal and NADPH oxidase-deficient neutrophils, arguing against a role of NADPH oxidase in spontaneous neutrophil apoptosis.

Neutrophil extracellular trap cell death requires both autophagy and superoxide generation

“…In contrast, we only observed a delay of apoptosis by stimulation with either LPS [26] or IL-8 (Supplementary information, Video S12). These results are in line with those of others who also did not observe NET formation in response to LPS [14].…”

“…These results further support our previous conclusion that ROS generation is insufficient to induce intracellular chromatin decondensation and subsequent NET formation. A nonsufficient role of ROS in NETosis is further supported by the inability of formyl-methionyl-leucyl-phenylalanine (fMLP, a potent inducer of Nox2 activity [26]) to induce NETosis (unpublished observations), presumably due to activation of signaling pathways known to inhibit apoptosis and autophagy [1,31,45]. In addition, stimulation of neonate neutrophils with PMA results in ROS production but not in NET formation, in contrast to PMA stimulation of adult neutrophils [21].…”

“…This pathway also eventually resulted in chromatin decondensation, but only after plasma membrane permeabilization during post-apoptotic secondary necrosis. Therefore, the chromatin decondensation that follows spontaneous apoptosis of neutrophils ex vivo [26] should not be con- Live cell images of neutrophils isolated from a CGD patient and stimulated with 100 nM PMA. Morphology was examined using DIC and chromatin decondensation using the cell-permeable DNA marker Hoechst 33342 (blue).…”

“…in NETosis, we used the PI3K inhibitor wortmannin, which inhibits autophagy [31,32]. Wortmannin treatment alone did not affect the kinetics ( Figure 4C) or morphological changes (Supplementary information, Video S6) of spontaneous neutrophil apoptosis at early time points, as reported previously [26]. As confirmed by live cell imaging, freshly isolated neutrophils pretreated with 100 nM wortmannin no longer show massive vacuolization on PMA stimulation, which further underscores the idea that the massive vacuolization is linked to autophagy induction ( Figure 4; Supplementary information, Video S7).…”

“…Cells were stimulated with 100 nM PMA and monitored every min for up to 300 min. volunteers, whether or not pretreated with DPI, die spontaneously from apoptosis [26], but more slowly than in PMA-induced NETosis (Figure 2A). Live cell imaging of the subcellular events described above revealed that fol- observed in CGD neutrophils at any time between PMA stimulation and plasma membrane disintegration ( Figure 2B and 2C).…”