TNFalpha-induced macrophage death via caspase-dependent and independent pathways

Abstract

Macrophages are the principal source of TNFalpha, yet they are highly resistant to TNFalpha-mediated cell death. Previously, employing in vitro differentiated human macrophages, we showed that following the inhibition of NF-kappaB, TNFalpha-induced caspase-8 activation contributes to DNA fragmentation but is not necessary for the loss of the inner mitochondrial transmembrane potential (DeltaPsim) or cell death. We here extend these observations to demonstrate that, when NF-kappaB is inhibited in macrophages, TNFalpha alters lysosomal membrane permeability (LMP). This results in the release of cathepsin B with subsequent loss of DeltaPsim and caspase-8 independent cell death. Interestingly, the cytoprotective, NF-kappaB-dependent protein A20 was rapidly induced in macrophages treated with TNFalpha. Ectopic expression of A20 in macrophages preserves LMP following treatment with TNFalpha, and as a result, mitochondrial integrity is safeguarded and macrophages are protected from cell death. These observations demonstrate that TNFalpha triggers both caspase 8-dependent and -independent cell death pathways in macrophages and identify a novel mechanism by which A20 protects these cells against both pathways.

Figure 1. Loss of lysosomal and mitochondrial integrity precedes DNA fragmentation or loss of cell membrane integrity

Differentiated macrophages were infected with Ad-IκBα (200 moi) or control vector and then incubated with TNFα (10 ng/ml) for the indicated time periods. Lysosomal integrity (defined by Lysotracker retention, A), loss of ΔΨm (% of control Rh123, B), apoptosis (<2N DNA, C), loss of cell membrane integrity (% PI+ cells, D), cytosolic cathepsin B activity (E) and cytosolic NAG activity (F) were analyzed. A–D, Results are the mean ± S.E. of 3 to 6 independent experiments, performed in duplicate. *, p < 0.01 or **, p < 0.005 versus control treatment. E–F, the values represent mean ± S.E. of an experiment performed in duplicate, which was representative of 2 independent experiments.

Figure 2. Cathepsin B contributes to TNFα-induced cell death

Differentiated macrophages were infected with Ad-IκB (200 moi) or a control adenoviral vector, followed by incubation with TNFα (10 ng/ml) for 24 hours. A–C, The cell permeable cathepsin B inhibitor CA-ME (2.5, 5 and 10 μM) was added to the cells 1 hour before TNFα. Loss of cell membrane integrity (A), < 2N DNA (B) and loss of mitochondrial membrane potential (C) were analyzed. D–F, Macrophages were transfected with cathepsin B siRNA (100nM, CatBi) or non-specific siRNA (NS). The effect of the siRNA on cathepsin B expression was examined by western blotting (D, panel on right). Following the addition of siRNA, the macrophages were infected with Ad-IκBα (200 moi) or the control vector, followed by incubation with TNFα (10 ng/ml) for 16 hours. The cells were then harvested and examined for the loss of cell membrane integrity (D), apoptosis (E) and ΔΨm (F). The results represent the mean ± S.E. of 3 independent experiments, performed in duplicate. * represents p < 0.01 or **, p < 0.005 versus control treatment.

Figure 3. Inhibition of both caspase-8 and cathepsin B protects against TNFα-induced cell death

Differentiated macrophages were infected with Ad-IκBα (50 moi, A–D; or 200 moi E). The cell membrane permeable caspase 8 selective inhibitor (IETD, 20 μM) or the cathepsin B inhibitor (CA-ME, 20 μM) were added to the cells 1 hour before the addition TNFα (10 ng/ml) for 16hours (AD) or 7 hours (E). The loss of cell membrane integrity (A), apoptosis (B), ΔΨm (C) and lysosomal integrity (D) were analyzed. The results represent the mean ± S.E. of 3 independent experiments, performed in triplicate. * represents p < 0.05 and ** < 0.01 versus control treatment. E, Cytosolic extracts were examined by cytochrome c released from the mitochondria by immunoblot analysis employing an anti-cytochrome c antibody. β-actin served as the loading control. The release of cytochrome c was compared with that observed following treatment with the control (DMSO), normalized to β-actin. The upper panel is representative of 2 independent experiments and the lower panel represents the mean ± S.E. of 2 independent experiments. * represents p < 0.05. F. Bid cleavage was examined by immunoblot analysis employing anti-Bid antibody. The cells were harvested 4 hours following the addition of TNFα. β-actin was used as the loading control. The experiment is representative of 2 independent experiments.

Figure 4. ROS is down stream regulator of lysosome pathway

Differentiated macrophages were infected with Ad IκBα (50 moi (A, B, C) or 200 moi (D, E)) followed by the addition of BHA (100 μM) and TNFα (10 ng/ml) for 16 hours (A, B, C) or 6–7 hours (D,E). The cells were then examined for loss of membrane integrity (A), DNA fragmentation (B), and mitochondrial inner membrane potential (C). The results are the mean ± S.E. of 2 independent experiments, performed in duplicate. D. Cytosolic cytochrome c was determined by western blot with anti-cytochrome c antibody. β-actin was used as control. EtOH was the vehicle for the BHA. The panel on the left is representative of 2 independent experiments. The panel on the right presents the mean ± S.E. of 2 independent experiments. The density of the cytochrome c bands was normalized with those for β-actin. E, Cytosolic cathepsin B activity. The results are the mean ± SE of 3 independent experiments, performed in duplicate. *, p < 0.05 versus control treatment.

Figure 5. The forced reduction of RIP only protects macrophages from DNA fragmentation

Differentiated macrophages were transfected with RIP siRNA (100nM, RIPi) or non-specific siRNA (NSi), and the expression of RIP repression was examined by western blotting (A). Cells were then infected with Ad-IκBα (50 moi) (A–D) or 200 moi (E,F) followed by incubation with TNFα (10 ng/ml) for 16 hours (B–D) or 4 hours (E, F), respectively. Loss of cell membrane integrity (D), DNA fragmentation (B), ΔΨm (C), caspase 8-like activity (E), and cytosolic cathepsin B (F) were determined. B–E. The results are the mean ± S.E. of 2 (F) or 3 (B–E) independent experiments, performed in triplicate. * represents p < 0.05 and ** p < 0.01 versus control treatment.

Figure 6. NF-κB-regulated A20 is rapidly induced by TNFα in macrophages

A. Differentiated macrophages were pretreated with TNFα (10 ng/ml) for 2 hours; the medium was then replaced with a new medium supplemented with PDTC (200 μM) plus TNFα (10ng/ml), followed by incubation for 6 to 24 hours. PBS and DMSO were used as the controls for TNFα and PDTC, respectively. Loss of cell viability was determined. The experiment was repeated twice with similar results. * represents p < 0.01 and ** p < 0.005 versus control treatment. B. differentiated macrophages were infected with Ad-IκBα (200 moi) or control adenoviral vector, followed by incubation with TNFα (10 ng/ml) or PBS for 2 hours. mRNA of TNFα-induced NF-κB-regulated genes were determined by microarray analysis. C. Macrophages were treated with TNFα (10 ng/ml) for the indicated times, after which the cells were harvested and the lysates examined by immunoblot analysis using an anti-A20 antibody.

Figure 7. A20 protects macrophages from TNFα-induced death

Differentiated macrophages were co-infected with Ad-IκBα or Ad-control vectors (50 moi) together with Ad-A20 (or the control) vectors (30 moi), followed by TNFα (10 ng/ml) for 16 hours (A, C–E) or for 4 hours (B, F). Lysosomal integrity (A), cytosolic cathepsin B activity (B), ΔΨm (C), cell viability (D), DNA fragmentation (E), and caspase-8-like activity (F) were determined. The results represent the mean ± S.E. of 3 independent experiments, performed in triplicate. **, p < 0.01 versus control treatment.