Anti-PR3 Antibodies Activate Oral Epithelial Cells Via PAR-2

Objectives: Anti-neutrophil cytoplasmic Abs targeting proteinase 3 (PR3) has been detected in relation to a wide range of inflammatory conditions such as periodontitis, and interaction of anti-PR3 Abs with endothelial and epithelial cells induces cell activation, although the underlying mechanism remains unclear. We previously demonstrated that PR3 activates human epithelial cells via protease-activated receptor (PAR)-2. In this study, we investigated whether oral epithelial cells express PR3 on the cell surface following stimulation with various proinflammatory cytokines, and whether anti-PR3 Abs activate oral epithelial cells expressing endogenous PR3 through the PAR-2 pathway. Methods: We examined PR3 mRNA in the cells by RT-PCR, PR3 expression on the cell surface by flow cytometry, PR3 protein in cell lysate and in the culture supernatant of the cells by western blotting, enzymatic activity of PR3 using PR3-specific substrate, and PR3 expression in human gingival tissues by immunohistochemistry. Results: Human oral epithelial cells expressed PR3 mRNA after treatment with proinflammatory cytokines such as IL-1alpha, TNF-alpha, IFN-alpha, -beta, and -gamma. A 29-kDa PR3 was expressed on the cell surface and released into culture supernatants stimulated with these cytokines. The addition of anti-PR3 Abs to cytokine-primed oral epithelial cells generated PR3 aggregation on the cells, and induced remarkable secretion of IL-8 and monocyte chemoattractant protein 1. RNA interference targeted to PAR-2 mRNA and intracellular Ca2+ mobilization assays demonstrated that anti-PR3 Abs activated epithelial cells through PAR-2. The anti-PR3 Abs-mediated cell activation was completely abolished by RNA interference targeted to PR3 mRNA and by inhibition of phospholipase C and NF-kappaB. Immunohistochemistry showed that inflamed oral epithelium actually expresses PR3 molecules. Conclusion: These findings suggest that oral epithelial cells express functional PR3 at the inflamed sites and respond to anti-PR3 Abs detected in diseased sera, and that these mechanisms may actively participate in inflammatory processes including periodontitis.