Tnf-α-accelerated apoptosis abrogates ANCA-mediated neutrophil respiratory burst by a caspase-dependent mechanism

Ralph Kettritz, Julia Scheumann, Y. X. Xu, Friedrich C. Luft, Hermann Haller, Shanying Lui, Lutz Jens, Uwe Heemann

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

Background. Tumor necrosis factor (TNF)-α rapidly primes neutrophils (PMN) for an antineutrophil cytoplasmic antibody (ANCA)-induced respiratory burst and is thus proinflammatory. TNF-α also progressively accelerates apoptosis. We investigated the effect of TNF-α-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-α or a combination of TNF-α and 2.5 μg/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-α 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-α 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-α-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-α exhibits dual actions by both priming and terminating ANCA-mediated activation of human PMN.

Original languageEnglish
Pages (from-to)502-515
Number of pages14
JournalKidney International
Volume61
Issue number2
DOIs
StatePublished - 2002
Externally publishedYes

Keywords

  • Anti-neutrophil cytoplasmic activity
  • PMN
  • Proinflammation
  • Respiratory burst
  • Tumor necrosis factor-α cell death

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