Abstract
During beneficial inflammation, potentially tissue-damaging granulocytes undergo apoptosis before being cleared by phagocytes in a non-phlogistic manner. Here we show that the rate of constitutive apoptosis in human neutrophils and eosinophils is greatly accelerated in both a rapid and concentration-dependent manner by the fungal metabolite gliotoxin, but not by its inactive analog methylthiogliotoxin. This induction of apoptosis was abolished by the caspase inhibitor zVAD-fmk, correlated with the inhibition of nuclear factor-kappa B (NF-kappaB), and was mimicked by a cell permeable inhibitory peptide of NF-kappaB, SN-50; other NF-kappaB inhibitors, curcumin and pyrrolidine dithiocarbamate; and the proteasome inhibitor, MG-132. Gliotoxin also augmented dramatically the early (2-6 h) pro-apoptotic effects of tumor necrosis factor-alpha (TNF-alpha) in neutrophils and unmasked the ability of TNF-alpha to induce eosinophil apoptosis. In neutrophils, TNF-alpha caused a gliotoxin-inhibitable activation of an inducible form of NF-kappaB, a response that may underlie the ability of TNF-alpha to delay apoptosis at later times (12-24 h) and limit its early killing effect. Furthermore, cycloheximide displayed a similar capacity to enhance TNF-alpha induced neutrophil apoptosis even at time points when cycloheximide alone had no pro-apoptotic effect, suggesting that NF-kappaB may regulate the production of protein(s) which protect neutrophils from the cytotoxic effects of TNF-alpha. These data shed light on the biochemical and molecular mechanisms regulating human granulocyte apoptosis and, in particular, indicate that the transcription factor NF-kappaB plays a crucial role in regulating the physiological cell death pathway in granulocytes.
Highlights
Neutrophilic and eosinophilic granulocytes originate from a common myeloid precursor; neutrophils are active in the defense against invading micro-organisms whereas eosinophils serve in anti-parasitic defenses and play a role in allergic inflammation
Induction of Apoptosis by Gliotoxin Is Dependent on Activation of the Caspase-cascade Pathway—We have recently demonstrated that the early pro-apoptotic effects of TNF-␣ in human neutrophils requires activation of both TNF-55 and TNF-75 receptor subtypes and thereby differs significantly from the priming effect of TNF-␣ which is signaled via the TNF-p55 receptor alone (10)
We have demonstrated that gliotoxin, but not its inactive derivative methylthiogliotoxin, (a) induces a direct time- and concentration-dependent increase in the rate of constitutive apoptosis in both neutrophils and eosinophils, (b) enhances the pro-apoptotic effect of TNF-␣ in neutrophils, and (c) reveals the cytotoxic effects of TNF-␣ in eosinophils
Summary
Neutrophils and eosinophils were isolated from the peripheral blood of normal donors by dextran sedimentation followed by centrifugation through discontinuous plasma-Percoll gradients (2, 29). Cells were mixed with washed 3G8-coated Dynabeads at a bead:neutrophil ratio of 3:1 on a rotary mixer at 4 °C for 20 min, and the beads removed magnetically by two 3-min stationary magnetic contacts (Dynal Magnetic Particle Concentrator, MPC-1) to yield an a eosinophil population of Ͼ98% purity. Cells were washed twice in phosphate-buffered saline without calcium and magnesium and once in phosphatebuffered saline before resuspending in Iscove’s DMEM (Life Technologies, Paisley, UK) with 10% autologous serum. Both cell types were cultured in flat-bottomed Falcon flexible wells (Becton Dickinson, Oxford, UK) at 37 °C in a 5% CO2 atmosphere; neutrophils at a concentration of 5 ϫ 106/ml and eosinophils at 2 ϫ 106/ml. All experiments were performed at least 3 times and each treatment done in triplicate
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