Tryptase increases proliferative activity of human conjunctival fibroblasts through protease-activated receptor-2.
OBJECTIVE
Tryptase that is released by mast cell degranulation has recently been thought to play a key role in wound healing in allergic bronchitis. Conjunctival fibroblasts secrete mediators and extracellular matrices that could exacerbate inflammation and papillary formation in allergic conjunctivitis. This study was conducted to investigate the effect of tryptase on the proliferation of conjunctival fibroblasts and studied whether this effect was mediated by protease-activated receptor (PAR)-2.


METHODS
Conjunctival fibroblasts were cultured with or without tryptase (0.1 ng/mL to 1.0 microg/mL), and the proliferation rate was assessed after 48 hours. The effects of tryptase inhibitors (leupeptin, benzamidine) and a PAR-2 agonist (SLIGKV) were examined. The existence of PAR-2 mRNA and protein in conjunctival fibroblasts was examined by RT-PCR and Western blot analysis, respectively. The existence of PAR-2 in cultured conjunctival fibroblasts and conjunctival papillae from patients with vernal keratoconjunctivitis, as well as conjunctival tissue from normal subjects was examined by immunohistochemistry.


RESULTS
Conjunctival fibroblast proliferation was upregulated by tryptase in a dose-dependent manner (P < 0.001). Leupeptin and benzamidine inhibited tryptase-induced fibroblast proliferation (P < 0.05), and SLIGKV mimicked tryptase's effect. PAR-2 mRNA and protein were detected in cultured conjunctival fibroblasts using RT-PCR and Western blot analysis. PAR-2 immunoreactivity in both the cultured conjunctival fibroblasts and in stromal cells in excised conjunctival tissues was observed.


CONCLUSIONS
Tryptase increased conjunctival fibroblast proliferation and this response appeared to be mediated by PAR-2. Mast cells are the most likely source of tryptase in the conjunctiva and may play an important role in chronic exacerbations with conjunctival papillary formation in allergic conjunctivitis.
Asano-Kato
Naoko
N
Department of Ophthalmology, School of Medicine, Keio University Shinanomachi, Tokyo, Japan. naokato@bc.iij4u.or.jp
Fukagawa
Kazumi
K
Okada
Naoko
N
Dogru
Murat
M
Tsubota
Kazuo
K
Fujishima
Hiroshi
H
D001550:Benzamidines / Q000494:pharmacology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007637:Keratoconjunctivitis / Q000378:metabolism / Q000473:pathology; D007976:Leupeptins / Q000494:pharmacology; D008407:Mast Cells / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D053802:Tryptases; D015854:Up-Regulation
D001550:Benzamidines; D007976:Leupeptins; D009842:Oligopeptides; D012333:RNA, Messenger; D044464:Receptor, PAR-2; D015842:Serine Proteinase Inhibitors; C109968:seryl-leucyl-isoleucyl--glycyl-lysyl-valine; D012697:Serine Endopeptidases; D053802:Tryptases; C032854:leupeptin; C032157:benzamidine