TNF-alpha--accelerated apoptosis abrogates ANCA-mediated neutrophil respiratory burst by a caspase-dependent mechanism.
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.
Kettritz
Ralph
R
HELIOS Klinikum-Berlin, Franz Volhard Clinic, and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Germany. kettritz@fvk-berlin.de
Scheumann
Julia
J
Xu
YaXin
Y
Luft
Friedrich C
FC
Haller
Hermann
H
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016897:Respiratory Burst / Q000187:drug effects / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D019268:Antibodies, Antineutrophil Cytoplasmic; D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; C430228:acetyl-aspartyl-glutamyl-valyl-lysine chloromethyl ketone; D003513:Cycloheximide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases