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

Abstract

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. 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. 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). 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.