Abstract
Phagocytosis of complement-opsonized targets is a primary function of neutrophils at sites of inflammation, and the clearance of neutrophils that have phagocytosed microbes is important for the resolution of inflammation. Our previous work suggests that phagocytosis leads to rapid neutrophil apoptosis that is inhibited by antibody to the beta2 integrin, Mac-1, and requires NADPH oxidase-derived reactive oxygen species (ROS) generated during phagocytosis. Here we report that phagocytosis-induced cell death (PICD) does not occur in Mac-1-deficient murine neutrophils, suggesting that PICD proceeds through a bona fide Mac-1-dependent pathway. A sustained, intracellular oxidative burst is associated with PICD. Furthermore, PICD does not require traditional death receptors, Fas, or tumor necrosis factor (TNF) receptor. TNF but not Fas synergizes with phagocytosis to enhance significantly PICD by increasing the oxidative burst, and this is Mac-1-dependent. Phagocytosis-induced ROS promote cleavage/activation of caspases 8 and 3, key players in most extrinsic ("death receptor") mediated pathways of apoptosis, and caspases 8 and 3 but not caspase 9/mitochondria, are required for PICD. This suggests that ROS target the extrinsic versus the intrinsic ("stress stimulus") apoptotic pathway. Phagocytosis also triggers a competing MAPK/ERK-dependent survival pathway that provides resistance to PICD likely by down-regulating caspase 8 activation. The anti-apoptotic factor granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly enhances ROS generation associated with phagocytosis. Despite this, it completely suppresses PICD by sustaining ERK activation and inhibiting caspase 8 activation in phagocytosing neutrophils. Together, these studies suggest that Mac-1-mediated phagocytosis promotes apoptosis through a caspase 8/3-dependent pathway that is modulated by NADPH oxidase-generated ROS and MAPK/ERK. Moreover, TNF and GM-CSF, likely encountered by phagocytosing neutrophils at inflammatory sites, exploit pro-(ROS) and anti-apoptotic (ERK) signals triggered by phagocytosis to promote or suppress PICD, respectively, and thus modulate the fate of phagocytosing neutrophils.
Highlights
Phagocytosis of complement-opsonized targets is a primary function of neutrophils at sites of inflammation, and the clearance of neutrophils that have phagocytosed microbes is important for the resolution of inflammation
Our previous work suggests that phagocytosis leads to rapid neutrophil apoptosis that is inhibited by antibody to the 2 integrin, Mac-1, and requires NADPH oxidase-derived reactive oxygen species (ROS) generated during phagocytosis
Materials and Preparation of FITC-labeled Yeast—Human tumor necrosis factor (TNF), TNF neutralizing antibody, and murine TNF were purchased from R&D Systems; Fas agonist antibody and Fas antagonist antibody were purchased from Upstate Biotechnology, Inc.; superoxide dismutase (SOD) and catalase were purchased from Roche Applied Science; Baker’s yeast, diphenyleneiodonium chloride (DPI), cytochalasin D, cycloheximide, luminol, isoluminol, LPS, and fMLP were from Sigma; FITC-labeled Escherichia coli was from Molecular Probes; and MEK inhibitors PD98059 and U0126 were from Promega
Summary
Materials and Preparation of FITC-labeled Yeast—Human TNF, TNF neutralizing antibody, and murine TNF were purchased from R&D Systems; Fas agonist antibody (clone CH11) and Fas antagonist antibody (clone ZB4) were purchased from Upstate Biotechnology, Inc.; superoxide dismutase (SOD) and catalase were purchased from Roche Applied Science; Baker’s yeast, diphenyleneiodonium chloride (DPI), cytochalasin D, cycloheximide, luminol, isoluminol, LPS, and fMLP were from Sigma; FITC-labeled Escherichia coli was from Molecular Probes; and MEK inhibitors PD98059 and U0126 were from Promega. Breast milk neutrophils, or bone marrow-derived mouse neutrophils (1 ϫ 106 cells per sample) were resuspended in DPBS (plus Ca2ϩ and Mg2ϩ) immediately prior to the following assays. Treatment of Neutrophils and Assessment of Apoptosis—Neutrophils (1 ϫ 106 cells) were incubated in DPBS (plus Ca2ϩ and Mg2ϩ) in the presence or absence of a variety of pro-apoptotic stimuli at 37 °C for 30 min These included human TNF (25 ng/ml) or murine TNF (30 ng/ml), Fas agonist antibody (100 ng/ml), as well as serum opsonized bioparticles (yeast or E. coli) (see above). At 2, 10, and 30 min after addition of yeast, ice-cold cell lysis buffer was added to aliquots of the samples for analysis of ERK activation. To confirm equivalent amount of loaded proteins, the membranes were stripped with 0.2 N NaOH for 5 min at room temperature and probed with ERK p42/p44 antibody (Santa Cruz Biotechnology)
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