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Evaluation of Caspase Activation to Assess Innate Immune Cell Death
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Evaluation of Caspase Activation to Assess Innate Immune Cell Death

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The activation of caspase-3 and DNA fragmentation in B cells phagocytosed by macrophages

Eiji Ninomiya1, Yuko Ito, Masa-Aki Shibata

  • 1Department of Anatomy and Biology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan.

Medical Electron Microscopy : Official Journal of the Clinical Electron Microscopy Society of Japan
|June 26, 2003
PubMed

Insights

Mammalian cell apoptosis involves two pathways. This study reveals that B cells in mouse gut-associated lymphoid tissues (GALTs) express cFLIP(L), blocking apoptosis until phagocytosis by macrophages triggers caspase activation and DNA fragmentation.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Biology

Background:

  • Mammalian cell apoptosis occurs via death receptor and mitochondrial pathways.
  • B cells in germinal centers (GCs) undergo differentiation into memory cells or plasma cells.
  • Distinguishing apoptotic B cells from differentiating B cells in GCs is challenging.

Purpose of the Study:

  • To investigate the apoptotic signaling pathways in B cells within mouse gut-associated lymphoid tissues (GALTs).
  • To determine the role of caspase family members and cFLIP(L) in B cell apoptosis within GCs.
  • To elucidate the sequence of events leading to B cell apoptosis in GALTs.

Main Methods:

  • Transmission electron microscopy (TEM) for ultrastructural analysis.
  • Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) assay for DNA fragmentation.
  • Immunofluorescence staining for caspases and cFLIP(L).
  • Caspase activity assays.

Main Results:

  • Isolated B cells in GCs expressed cFLIP(L) but lacked active caspase-3 and TUNEL immunoreactivity.
  • Phagocytosed B cells exhibited active caspase-3 and TUNEL signals, with absent cFLIP(L).
  • Caspase activity assays showed high activity of caspase-3, caspase-9, and caspase-8 in GALTs.

Conclusions:

  • cFLIP(L) expression likely inhibits the death receptor pathway in GALTs B cells.
  • Caspase-3 activation and DNA fragmentation are initiated upon macrophage phagocytosis of B cells.
  • Apoptotic signaling in GALTs B cells is tightly regulated and occurs post-phagocytosis.

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