Role Of NF-κB In The Regulation Of Non-apoptotic, Non-necrotic Neutrophil Death Induced By ONO-AE-248

Objective: Polymorphonuclear neutrophils (PMNs) are the most important effector cells in inflammation. The recruitment of many hyperactive PMNs at an inflammatory site may cause secondary tissue injury and contribute to neutrophil-mediated diseases. PMNs, terminally differentiated cells, undergo spontaneous apoptosis in vitro and in vivo, and this process has been recognized as a crucial mechanism for accomplishing resolution of inflammation and maintaining the stability of internal environment. During the study of apoptosis, an increasing number of reports have suggested that apoptosis is not exclusive form of programmed cell death (PCD). Our previous study has showed that ONO-AE-248, a selective EP3 receptor agonist, rapidly caused a novel form of neutrophil death resembling neither typical apoptosis nor necrosis. The unique neutrophil death is independent of the activity of caspase-3, caspase-8, and phosphorylation of p38-MAPK. To date, the molecular mechanism of this new type of cell death has not been well defined, yet nuclear factor kappa B (NF-κB) is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. Thus, we investigated whether activation of NF-κB signaling pathway contributes to the ONO-AE- 248-induced neutrophil death. Methods: Heparinized peripheral blood was obtained from healthy volunteers. Neutrophils were isolated using 3% dextran sedimentation followed by density gradient centrifugation with Ficoll. The purity of neutrophil population was > 96% on the Wright- Giemsa stain, and neutrophil viability was > 96%, as determined by trypan blue dye exclusion. The neutrophils were cultured in the presence or absence of ONO-AE-248(5×10-5 M)for the indicated time periods. Apoptosis of neutrophils was detected by flow cytometric analysis of the nucleus. Low-molecular-weight DNA was isolated from the neutrophils and was detected by agarose gel electrophoresis. NF-κB activation was examined by Western blotting analysis. Expression levels of NF-κB-regulated gene products, including X-linked inhibitory apoptosis protein (XIAP), defender against apoptotic death (DAD1), FLICE inhibitory protein (FLIP) and DNA fragmentation factor 40 (DFF40), were detected by RT-PCR. Results: Flow cytometry suggested that neutrophils cultured with medium alone exhibited a hypodiploid DNA peak, which is a characteristic feature of apoptosis, but not in ONO-AE-248-treated neutrophils. DNA fragmentation examined by agarose gel electrophoresis of DNA was not apparent in ONO-AE- 248-stimulated neutrophils. Western blotting and/or RT-PCR showed that ONO-AE-248 did not affect IκBαdegradation but caused the down-regulation of DFF40 expression. Conclusion: ONO-AE-248 could induce a form of non-apoptotic, non-necrotic cell death of neutrophils in vitro, which is independent of NF-κB signaling pathway. In addition, our experiments indicate that the down-regulation of DFF40 expression is likely to be one of explanations for the lack of the oligonucleosomal DNA fragmentation in the ONO-AE-248-induced neutrophil death. Taken together, these results reveal that spontaneous apoptosis of neutrophil is probably not only way of its resolution.