title: Regulation of apoptosis of myeloid immune cells: implication for cancer therapy and inflammation
Creators: name: Shehu, S
Publisher: name: University of Liverpool Repository
Year: 2017

description: Objectives: This project was undertaken to investigate the potential of the human PLB-985 cell line to terminally differentiate, along the myeloid lineage into mature neutrophil-like granulocytes, and to identify the molecular properties of the differentiated PLB-985 cells in comparison with blood neutrophils, in order to establish their usefulness as model of neutrophil differentiation and functions. Methods: Human neutrophils were isolated from whole blood of healthy, consented, adult donors. The human PLB-985 cells were grown in routine culture media of RPMI 1640 (+10mM L-glutamine). Exponentially growing PLB-985 cells were induced to differentiate into neutrophil-like cells in differentiation culture media of RPMI 1640 (+10mM L-glutamine), supplemented with three differentiation and maturation-inducing agents: ATRA, DMF and sodium pyruvate. Granulocytic differentiation was measured by cell morphology using light microscope, apoptosis was determined by morphology or flow cytometry, cell viability, cell cycle parameters, expression of cell surface receptors and phagocytosis were determined by flow cytometry, oxidative burst activity was measured using flow cytometry and luminol-enhanced chemiluminescence assay, and expression of apoptotic proteins was determined by western blotting. Results: A modified differentiation protocol and optimisation procedure has been established. Terminally-differentiated, neutrophil-like PLB-985 cells, have been consistently cultured, that resemble mature blood neutrophil morphology, evident by the acquisition of multi-lobed nucleus and granulated cytoplasm, and which had an appreciably extended lifespan. It was discovered that supplementing the differentiation medium with either G-CSF or GM-CSF both improved differentiation and delayed apoptosis of the differentiated cells. These cells underwent cell cycle arrest and progression into apoptosis and they resembled mature blood neutrophils in terms of cell cycle parameters and apoptotic morphology. The differentiation induced expression of anti-apoptotic proteins, Mcl-1 and Bcl-XL in these cells and their expression levels correlated with cell survival status, whilst Bcl-2 expression was lost. The differentiated PLB-985 cells also displayed phagocytosis and oxidative burst activity, expressed cell surface receptors for CD11b, CD14 and CD16 and expressed strong chemotactic transmigration towards chemoattractants. Finally, JAK inhibitors baricitinib and tofacitinib suppressed the growth, differentiation and viability of the differentiated cells, increased their progression into apoptosis, and down-regulate their responses to G-CSF and GM-CSF signalling. Hence, they may inhibit in vitro granulopoiesis, and may have anti-inflammatory activity. Conclusions: This study has indicated that under appropriate conditions, PLB-985 cells can differentiate terminally into mature neutrophil-like phenotypes that resemble blood neutrophils morphologically and functionally, with an enhanced survival of the mature cells. These differentiated PLB-985 cells may therefore, provide an excellent neutrophil-model system for in vitro study of neutrophil differentiation and functions, such as apoptosis regulation.