Promyelocyte HL-60 Cell-line Demonstrates the Involvement of NETosis-Related Proteins in Neutrophil Nuclear Morphology Dynamics
Neutrophils are characterized by a unique nuclear morphology, where the nucleus possesses a segmented multi-lobed form. During inflammation, neutrophils become activated and utilize their anti-microbial abilities. Neutrophil activation and extracellular trap formation are associated with the cell undergoing a change in nuclear morphology via delobing of the nucleus. While much work has been done concerning which parts of the activation pathway are required for NETosis, little is known about which portions of the activation pathway are involved with these morphology dynamics. Using quantitative image analysis, we established the promyelocyte HL-60 cell-line as a model system in which to study human neutrophil nuclear morphology dynamics and differentiated HL-60s as a model to investigate activation-induced nuclear morphological changes. We find that ATRA differentiated HL-60s and DMF differentiated HL-60s can be used to observe changes in the measured circularity of the nucleus induced by PMA and A23187 stimulation respectively. Using chemical inhibitors in differentiated HL-60 stimulation, it was discovered that NOX and NE activity are required for PMA-induced nuclear morphological change while MPO and PAD4 activity are not, and that PAD4 activity is required for A23187-induced nuclear morphological change. These findings demonstrate how factors that are known to contribute to NETosis also contribute to pre-NETosis neutrophil nuclear dynamics.