TNF-alpha--accelerated apoptosis abrogates ANCA-mediated neutrophil respiratory burst by a caspase-dependent mechanism

Kidney Int. 2002 Feb;61(2):502-15. doi: 10.1046/j.1523-1755.2002.00161.x.


Background: Tumor necrosis factor (TNF)-alpha rapidly primes neutrophils (PMN) for an anti-neutrophil cytoplasmic antibody (ANCA)-induced respiratory burst and is thus proinflammatory. TNF-alpha also progressively accelerates apoptosis. We investigated the effect of TNF-alpha-mediated apoptosis on ANCA antigen expression and on ANCA-induced superoxide generation in human PMN.

Methods: PMN were brought to apoptosis by 10 ng/mL of TNF-alpha or a combination of TNF-alpha and 2.5 microg/mL cycloheximide, a protein synthesis inhibitor, or cycloheximide alone for three hours. Apoptosis and ANCA antigen expression were assessed by fluorescence-activated cell sorting (FACS) and microscopy. Superoxide was determined with the ferricytochrome C assay.

Results: TNF-alpha with cycloheximide for three hours caused apoptosis in 87% PMN compared to 2% in untreated controls (N=18; P < 0.01). Accelerated apoptosis was associated with an increase in ANCA-antigen expression for both proteinase 3 and myeloperoxidase (P < 0.05). Nevertheless, apoptosis was paralleled by a decreased proteinase 3 and myeloperoxidase ANCA-induced respiratory burst (P < 0.05). Furthermore, superoxide release in response to immune complexes, phorbol ester (PMA), and bacterial peptide (FMLP) was significantly decreased. Blocking caspase-3 activity prevented apoptosis in TNF-alpha with cycloheximide-treated cells (83% to 2%) and prevented compromised respiratory burst in response to ANCA. Caspase-3 inhibition abrogated apoptosis-mediated ANCA antigen up-regulation (PR3 141.6 +/- 34.1 MFI to 33.9 +/- 7.8; MPO 48.3 +/- 12.9 MFI to 11.9 +/- 3.2, N=6, P < 0.05).

Conclusions: TNF-alpha-accelerated apoptosis was associated with increased ANCA antigen expression but with down-regulated respiratory burst activity in response to ANCA. Specific inhibition of apoptosis by caspase-3 blockade prevented the increase in ANCA-antigen expression and preserved the capability of generating superoxide, thereby establishing a causative role for apoptosis. We suggest that TNF-alpha exhibits dual actions by both priming and terminating ANCA-mediated activation of human PMN.

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology
  • Antibodies, Antineutrophil Cytoplasmic / pharmacology*
  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects*
  • Apoptosis / immunology
  • Caspase 3
  • Caspase Inhibitors
  • Caspases / metabolism*
  • Cell Membrane / metabolism
  • Cycloheximide / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Flow Cytometry
  • Humans
  • In Vitro Techniques
  • Neutrophils / cytology*
  • Neutrophils / drug effects
  • Neutrophils / immunology
  • Protein Synthesis Inhibitors / pharmacology
  • Respiratory Burst / drug effects
  • Respiratory Burst / immunology
  • Tumor Necrosis Factor-alpha / pharmacology*


  • Amino Acid Chloromethyl Ketones
  • Antibodies, Antineutrophil Cytoplasmic
  • Antineoplastic Agents
  • Caspase Inhibitors
  • Enzyme Inhibitors
  • Protein Synthesis Inhibitors
  • Tumor Necrosis Factor-alpha
  • acetyl-aspartyl-glutamyl-valyl-lysine chloromethyl ketone
  • Cycloheximide
  • CASP3 protein, human
  • Caspase 3
  • Caspases