Apoptosis of endothelial cells induced by the neutrophil serine proteases proteinase 3 and elastase.
The pathogenesis of vasculitis associated with anti-neutrophil cytoplasmic antibodies is not established. The anti-neutrophil cytoplasmic antibody autoanigens proteinase 3 (PR3) and elastase induce detachment and cytolysis of endothelial cells in vitro. We investigated whether PR3 and elastase trigger endothelial cell apoptosis. Primary bovine pulmonary artery endothelial cells were treated with either PR3, elastase, or myeloperoxidase (MPO) and apoptosis assessed by four different methods. By the cell death detection enzyme-linked immunosorbent assay, DNA fragmentation increased to 208 +/- 84% or 153 +/- 27% of control with 1 micrograms/ml PR3 or elastase at 24 hours. By ultraviolet light microscopy, the percentage of apoptotic cells significantly increased (P < 0.05) with 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase at 6, 12, or 24 hours. Values at the 24-hour time point are 15.3 +/- 6.4% or 25.8 +/- 6.6% for 5 or 10 micrograms/ml PR3 and 13.9 +/- 3.6% or 20.7 +/- 1.8% for 25 or 50 micrograms/ml elastase compared with 2.2 +/- 1.2% for control. Similarly, with flow cytometry, 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase for 6, 12, or 24 hours demonstrated increasing apoptosis in a dose- and time-dependent manner with the highest values achieved at 24 hours (23.4 +/- 4.0% and 35.6% for 5 and 10 micrograms/ml PR3 and 31.8 +/- 4.0% and 47.8% for 25 and 50 micrograms/ml elastase compared with 7.9 +/- 2.2% in control). Typical DNA laddering was apparent from 6 to 24 hours at 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase. Myeloperoxidase did not induce cell apoptosis. Release of PR3 and elastase by activated neutrophils during acute inflammation, including anti-neutrophil cytoplasmic antibody-associated vasculitis, may result in vascular damage by endothelial cell apoptosis.
Department of Medicine, University of North Carolina at Chapel Hill 27599-7155, USA.
J J
JJ
Yang
R
R
Kettritz
R J
RJ
Falk
J C
JC
Jennette
M L
ML
Gaido
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002417:Cattle; D002478:Cells, Cultured; D004587:Electrophoresis, Agar Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D019272:Leukocyte Elastase / Q000494:pharmacology*; D008853:Microscopy; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology*; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects; D012697:Serine Endopeptidases / Q000633:toxicity*; D014466:Ultraviolet Rays
D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D053146:Myeloblastin