Tryptase Increases Conjunctival Fibroblast Proliferation via Protease–Activated Receptor–2

Abstract

Abstract: : Purpose:Mast cell is one of main effector cells for allergic conjunctivitis. Tryptase, a proteolytic enzyme in mast cell secretory granules, has recently been reported to be one of the key factors for wound healing in allergic bronchitis. Conjunctival fibroblast proliferation is thought to involve papillary formation in allergic conjunctivitis. We investigated the effect of tryptase on proliferation of conjunctival fibroblasts and whether the effect was mediated by a tryptase receptor, protease–activated receptor–2 (PAR–2). Methods:Conjunctival fibroblasts were cultured in DMEM/F12 medium with 10 FCS for 24 hours. Then, culture medium was replaced to FCS free DMEM/F12 with or without tryptase (0.1, 1.0, 10, 100 ng/ml, and 1.0µg/ml), and proliferation rate was assessed by WST assay after 48 hours. The effects of tryptase inhibitors (leupeptin and benzamidine) and PAR–2 agonist (SLIGKV) were also examined. Existence of mRNA and protein of PAR–2 on conjunctival fibroblasts were examined by RT–PCR and western blotting, respectively. The existence of PAR–2 was also examined by immunohistochemistry on cultured conjunctival fibroblasts and surgically excised conjunctival papillae from patients with VKC. Results:Conjunctival fibroblast proliferation was upregulated by tryptase in a dose–dependent manner (p<0.001). Leupeptin and bezamidine inihibited the tryptase–induced fibroblast proliferation (p<0.05), and SLIGKV mimicked tryptase’s effect. The results of RT–PCR and western blotting showed PAR–2 mRNA and protein in cultured conjunctival fibroblasts. PAR–2 immunoreactivity was positive in cytoplasm of cultured conjunctival fibroblasts and also in fibroblasts within conjunctival papillae. Conclusions:The present results showed that tryptase increased conjunctival fibroblast proliferation and this response was mediated by PAR–2. Mast cells may play an important role through tryptase not only in early phase reactions but also in chronic exacerbation of conjunctival papillary formation in allergic conjunctivitis.

Keywords: conjunctivitis • inflammation • proliferation