Augmented intracellular glutathione inhibits fas-triggered apoptosis of activated human neutrophils

Q1Q1Agonist signals delivered through cell surface Fas induce apoptosis. However, the apoptotic program can be modulated by signals from the environment, and in particular, by signals delivered through adhesion molecules. Because neutrophil functional activity in inflammation is contingent on cell survival, and because circulating neutrophils normally die rapidly through a constitutively expressed apoptotic program, we evaluated Fas-mediated apoptosis in resting and inflammatory human neutrophils. We show that normal neutrophils respond to Fas engagement with accelerated rates of apoptosis, but cross-linking of β2 integrins or priming with bacterial lipopolysaccharide (LPS) prevents this increase. Adhesion molecule cross-linking results in increased intracellular glutathione (GSH). Augmentation of intracellular GSH with exogenous GSH or N-acetylcysteine is sufficient to reduce the Fas-triggered increase in apoptotic rates. Prevention of the activation induced GSH increase by buthionine sulfoximine, a cell permeable inhibitor of GSH biosynthesis, restored Fas responsiveness in activated neutrophils, an effect that could be blocked with exogenous GSH. Taken together, these data show that Fas-induced signaling for neutrophil apoptosis is blocked in a redox sensitive manner by costimulatory signals delivered through β2 integrins or activation by LPS, and provide a biologic explanation for sustained neutrophil survival in the inflammatory environment.Revista Nacional - Indexad

Regulation of apoptosis and priming of neutrophil oxidative burst by diisopropyl fluorophosphate

Abstract Background Diisopropyl fluorophosphate (DFP) is a serine protease inhibitor that is widely used as an inhibitor of endogenous proteases in in vitro neutrophil studies. Its effects on neutrophil function are unclear. We sought to determine the biological effects of DFP on human neutrophil apoptosis and oxidative burst. Methods We isolated neutrophils from healthy volunteers, incubated them with DFP (2.5 mM), and evaluated neutrophil elastase (NE) activity, neutrophil degranulation, apoptosis as reflected in hypodiploid DNA formation and exteriorization of phosphatidylserine (PS), processing and activity of caspases-3 and -8, oxidative burst activity and hydrogen peroxide release. Results Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules. DFP inhibited constitutive neutrophil apoptosis as reflected in DNA fragmentation, and the processing and activity of caspases-3 and -8. DFP also inhibited priming of neutrophils for oxidative burst activity and hydrogen peroxide release. However, DFP enhanced the exteriorization of PS in a dose-dependent manner. Conclusion We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity. We further conclude that endogenous proteases play a role in the biology of constitutive neutrophil apoptosis

Pre–B cell colony–enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis

Pre–B cell colony-enhancing factor (PBEF) is a highly conserved 52-kDa protein, originally identified as a growth factor for early stage B cells. We show here that PBEF is also upregulated in neutrophils by IL-1β and functions as a novel inhibitor of apoptosis in response to a variety of inflammatory stimuli. Induction of PBEF occurs 5–10 hours after LPS exposure. Prevention of PBEF translation with an antisense oligonucleotide completely abrogates the inhibitory effects of LPS, IL-1, GM-CSF, IL-8, and TNF-α on neutrophil apoptosis. Immunoreactive PBEF is detectable in culture supernatants from LPS-stimulated neutrophils, and a recombinant PBEF fusion protein inhibits neutrophil apoptosis. PBEF is also expressed in neutrophils from critically ill patients with sepsis in whom rates of apoptosis are profoundly delayed. Expression occurs at higher levels than those seen in experimental inflammation, and a PBEF antisense oligonucleotide significantly restores the normal kinetics of apoptosis in septic polymorphonuclear neutrophils. Inhibition of apoptosis by PBEF is associated with reduced activity of caspases-8 and -3, but not caspase-9. These data identify PBEF as a novel inflammatory cytokine that plays a requisite role in the delayed neutrophil apoptosis of clinical and experimental sepsis

Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src.

Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival

Tyrosine phosphorylation of caspase-8A in multiple sites follows Src expression.

<p>A) Caspase-8A structure and SFK tyrosine phosphorylation sites predicted by GPS2.1. B) HEK293 cells were co-transfected with inactive C377S mutant of GFP-caspase-8A and Y527F Src for 24 hours then lysates immunoprecipitated using an anti-GFP antibody. GFP-caspase-8A IP was subjected to SDS-PAGE, and the appropriate band (80 kDa) was excised and sent for LC-MS/MS analysis. C) Mutagenesis was performed at Y397/Y380 and Y465/Y448 sites to generate phosphomimetic (tyrosine to glutamic acid) and non-phosphorylatable (tyrosine to phenylalanine) caspase-8A/B mutants.</p

Positive feedback loop of caspase-8A induced Src activation.

<p>Phosphorylation of caspase-8A by Src at Y465 prevents its cleavage, and so prevents the induction of apoptosis. The presence of inactive caspase-8A (phosphorylated at Y465) allows the phosphorylation of Y397. When Y397 is phosphorylated in the presence of Y465 phosphorylation, caspase-8A then becomes a Src activator, by binding to the SH2 domain of Src via its pY₃₉₇LEM peptide, further activating Src by promoting Y416 phosphorylation of Src.</p

Y465 phosphomimetic modification of caspase-8A abolishes its ability to activate caspase-3 and to induce apoptosis.

<p>WT GFP-caspase-8A mutants were transfected into HEK293 cells. A) Cells were fixed and immunostained with anti-cleaved caspase-3 antibody. Green represents GFP-caspase-8A expressing cells; orange represents active caspase-3 in GFP-caspase-8A expressing cells (white arrow). B) The percentage of active caspase-3 positive cells is displayed. N = 3, * <i>p</i> < 0.05. C) Cell morphology of transfected cells was examined using live fluorescence microscopy. Healthy cells are identified with orange arrows whereas apoptotic cells characterized by cell rounding or apoptotic bodies are identified with white arrows. D) Transfected cells were permeabilized and stained with propidium iodide to qualified the percentage of cells expressing hypodiploid DNA representing DNA fragmentation seen in apoptosis. N = 3, <i>p</i> < 0.05.</p

Inhibition of caspase-8 activity enhances Src-dependent tyrosine phosphorylation of caspase-8.

<p>A) HEK293 cells were transfected with GFP-caspase-8A (WT or C377S inactive mutant) with or without Y527F Src for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody and anti-GFP antibody. B) HEK293 cells were transfected with GFP-caspase-8A (WT or C377S inactive mutant) with Y527F Src for 24 hours. GFP-caspase-8A was immunoprecipitated with anti-GFP antibody and precipitates were probed with anti-phospho-tyrosine antibody and anti-GFP antibody. C) HEK293 cells were transfected with WT GFP-caspase-8A and Y527F Src for 9 hours followed by treatment with DMSO vehicle control or caspase-8 inhibitor (20 μM) for 15 hours. Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody and anti-GFP antibody.</p