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Apoptosis by pan-caspase inhibitors in lipopolysaccharide-activated macrophages
1Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
Abstract:
Although apoptosis has been observed in macrophages during the course of infections, the mechanism of apoptosis in activated macrophages is not fully understood. This study shows that pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) or t-butyloxycarbonyl-Asp-fluoromethylketone (Boc-D) caused the death of lipopolysaccharide (LPS)-activated macrophages and RAW 264.7 cells with apoptotic features. The apoptosis was also observed in lipoprotein-treated bacteria but not in CpG oligonucleotide- or flagellin-treated macrophages, indicating a difference of cellular responses downstream of different Toll-like receptors. Consistent with the induction of cell death by pan-caspase inhibitors, no activation of known caspases was detected in LPS-ZVAD-treated cells, suggesting an involvement of unknown proapoptotic caspases in the cell death. ZVAD inhibited the activation of extracellular signal-regulated kinase (ERK) and p38 but not of nuclear factor (NF)-kappa B induced by LPS, suggesting that the ZVAD-sensitive molecule lies upstream of the ERK and p38 pathways but downstream of the divergent site of NF-kappa B and mitogen-activated protein kinases. Our results demonstrate that apoptosis of macrophages induced by LPS+ZVAD is independent from the known proapoptotic caspases and suggest that activity of an unidentified ZVAD-sensitive molecule(s) is involved in the survival of LPS-activated macrophages.
Insights
Pan-caspase inhibitors induce apoptosis in activated macrophages, revealing an unknown cell death pathway. This suggests a novel ZVAD-sensitive molecule is crucial for macrophage survival during infections.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Apoptosis in macrophages during infection is known but not fully understood.
- Activated macrophages play critical roles in immune responses.
Purpose of the Study:
- To elucidate the mechanism of apoptosis in activated macrophages.
- To identify novel pathways involved in macrophage cell death.
Main Methods:
- Treatment of macrophages and RAW 264.7 cells with lipopolysaccharide (LPS) and pan-caspase inhibitors (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone [ZVAD] or t-butyloxycarbonyl-Asp-fluoromethylketone [Boc-D]).
- Analysis of cellular responses to various Toll-like receptor (TLR) agonists.
- Investigation of caspase activation, extracellular signal-regulated kinase (ERK), p38, and nuclear factor-kappa B (NF-kappa B) pathways.
Main Results:
- ZVAD and Boc-D induced apoptosis in LPS-activated macrophages and RAW 264.7 cells.
- Apoptosis was observed with lipoprotein-treated bacteria but not with CpG or flagellin, indicating TLR-dependent differences.
- No activation of known caspases was detected in LPS-ZVAD-treated cells.
- ZVAD inhibited LPS-induced ERK and p38 activation but not NF-kappa B activation.
Conclusions:
- LPS+ZVAD-induced macrophage apoptosis is independent of known proapoptotic caspases.
- An unidentified ZVAD-sensitive molecule(s) likely plays a role in the survival of LPS-activated macrophages.
- This study uncovers a novel caspase-independent apoptotic pathway in macrophages.