NADPH Oxidase-dependent Oxidation and Externalization of Phosphatidylserine during Apoptosis in Me2SO-differentiated HL-60 Cells

Abstract

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Neutrophils aid host defense by killing invading microorganisms through production of highly reactive oxygen species (ROS) 1 generated by activation of the NADPH oxidase complex. When released inappropriately into the extracellular milieu, these ROS can cause persistent inflammation and considerable damage to the surrounding, healthy tissues. To prevent calamitous release of ROS, macrophages remove excess activated neutrophils from an inflammatory site in a regulated way, through processes that ensure swift resolution of inflammation yet make provision for neutrophils to fulfill their microbicidal function. Phagocytic cells carry out this clearance by recogniz-

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me’ signals: cleavage and inhibition of phagocytosis by Lp-PLA2

“…Our previous work has emphasized pro-phagocytic effects of externalized PSox species 9 and the role of NADPH oxidase in apoptotic cell clearance. 11 Subsequently, Greenberg et al 8 employed myeloperoxidase/glucose oxidase oxidation in model membranes composed of oxidizable PS in palmitoyloleyl-PC (carrier) and demonstrated PSox-dependent phagocytosis of liposomes and apoptotic cells by macrophages. 8 The importance of unidentified 'pro-oxidant' factors has been also noted in regulatory effects of lyso-PS on phagocytosis of neutrophils.…”

“…7,8 This concept has gained support due to detection in apoptotic cells of different oxidized species of PS. [8][9][10][11] More recently, other PS metabolites such as lyso-PS have also been identified to promote engulfment by binding G2A receptors on phagocytes. 12,13 Another paradox with the concept that PS is the universal 'eat-me' signal comes from observations that, during normal physiologic activation, several cell types including macrophages, lymphocytes and endothelial cells externalize PS at levels sufficient for detection, yet escape engulfment.…”

Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

“…Baker BV, Deventer, The Netherlands). 51 Fresh medium with DMSO was added every other day of culture to prevent cell overgrowth.…”

Milk fat globule epidermal growth factor 8 (MFG-E8) binds to oxidized phosphatidylserine: implications for macrophage clearance of apoptotic cells

“…We have previously documented the occurrence of cell surface externalization of oxidized PS (PS-OX) during apoptosis, and have provided evidence that both species of PS (oxidized and nonoxidized) function as recognition signals for macrophages. 5,6 We now show that MFG-E8 preferentially interacts with PS-OX and, to a lesser degree, with nonoxidized PS and PC (phosphatidylcholine).…”