Anti-PR-3 antibodies induce endothelial IL-8 release.
BACKGROUND
It has been shown that interaction of anti-PR-3 antibodies with human endothelial cells (EC) leads to an activation of EC in vitro, i.e. induction of adhesion molecules like E-selectin, VCAM-1 and tissue factor. The aim of this study was to investigate the effect of anti-PR-3 antibodies on endothelial IL-8 expression.


METHODS
EC were cultured in 96-well plates and stimulated with TNF-alpha and IL-1beta for 1 h to induce membrane expression of endothelial PR-3. Anti-PR-3 antibodies were purified from sera from patients with clinically active Wegener's granulomatosis. Purified anti-Ro, anti-centromere, anti-dsDNA antibodies and a monoclonal anti-PR-3 antibody (WGM2) served as controls. Induction of IL-8 mRNA was detected by RT-PCR. IL-8 was measured in the supernatant of EC by ELISA. In addition, induction of NFkappaB was investigated by PAGE of nuclear extracts of EC and Western blot with ab against p65.


RESULTS
In contrast to controls, interaction of anti-PR-3 antibodies (patients and WGM2) with cytokine activated EC led to the highest amount of IL-8 synthesis. Priming of EC with cytokines alone induced a markedly lower IL-8 level. The lowest levels of IL-8 could be measured after incubation of unprimed EC with anti-PR-3 antibodies. Anti-PR-3 antibody induced endothelial IL-8 expression could be inhibited by cycloheximide. In addition, we established that the activation of NF-kappaB is critically involved in anti-PR-3 antibody induced endothelial IL-8 production.


CONCLUSIONS
In summary, we were able to show that anti-PR-3 antibodies induce endothelial IL-8 synthesis by activating NF-kappaB. As IL-8 represents a powerful neutrophil chemoattractant, our data provide further evidence for a direct pathogenic effect of anti-PR-3 antibodies in ANCA related diseases.
Mayet
W
W
First Medical Department, University of Mainz, Germany mayet@mail.uni-mainz.de
Schwarting
A
A
Barreiros
A P
AP
Schlaak
J
J
Neurath
M
M
D019268:Antibodies, Antineutrophil Cytoplasmic / Q000097:blood; D001323:Autoantibodies / Q000276:immunology* / Q000494:pharmacology; D001324:Autoantigens / Q000276:immunology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology / Q000276:immunology*; D014890:Granulomatosis with Polyangiitis / Q000097:blood / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D053146:Myeloblastin; D016328:NF-kappa B / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000235:genetics / Q000276:immunology*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins
D019268:Antibodies, Antineutrophil Cytoplasmic; D001323:Autoantibodies; D001324:Autoantigens; D007074:Immunoglobulin G; D007375:Interleukin-1; D016209:Interleukin-8; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D012697:Serine Endopeptidases; D053146:Myeloblastin