This confirms that both PARs are expressed and are functional. It has been shown previously that both trypsin and tryptase at high concentrations may cleave PAR1 in vitro, in addition to PAR2 . when incubated with specific PAR-activating enzymes or agonist peptides. Thrombin- and tryptase-induced HCEC activation was blocked by PAR1 and PAR2 neutralizing antibodies, respectively, and by specific enzyme inhibitors. The constitutive expression of PAR1 and PAR2, and their activation by thrombin and tryptase, respectively, may have important implications in ocular inflammation. INTRODUCTION Allergic inflammation of the ocular conjunctiva is usually associated with increased mast cell mediators in tear fluid, and the recruitment of activated eosinophils, mast cells, and lymphocytes . Furthermore, ocular epithelial cells are active participants in the regulation of allergic inflammation via expression of adhesion molecules and elaboration of proinflammatory cytokines and chemokines . Recent work has highlighted the potential role of protease-activated receptors (PARs) in stimulating cytokine MK-6096 (Filorexant) production by respiratory epithelium . In addition, Lang et al  have demonstrated the presence of PAR1 and PAR2 in the corneal epithelium. However, the expression and functions of PAR1 and PAR2 in bulbar conjunctival epithelium have not been explored. PARs are G-protein-coupled seven transmembrane receptors [5, 6] with a unique signaling mechanism. These receptors have their own ligands embedded in their extracellular are increased in a variety of atopic ocular disorders . Finally, PAR2 is usually upregulated in the respiratory epithelia of patients with asthma . The results presented in the report show that PAR1 and PAR2 mRNA and proteins are constitutively expressed in HCECs and their stimulation by thrombin and tryptase, respectively, results in the release of IL-6. METHODS Materials Human conjunctival epithelial cells (HCECs, Wong Kilborne derivative of Chang epithelial cells) were obtained from American Tissue Type Culture Collection. Epithelial cell growth medium with The cultured HCECs were fixed with paraformaldehyde and were incubated with specific primary antibodies or isotype IgG. After extensive washing, the cells were incubated with rhodamine-conjugated secondary antibodies and viewed under a fluorescence microscope for imaging. Functional activity of PAR1 and PAR2 Previous reports document that thrombin induces IL-6 production by human umbilical vein endothelial cells  and bronchial epithelial cells . In addition, trypsin induces cytokine production in corneal  and bronchial epithelial cells . MK-6096 (Filorexant) Therefore, to determine the functional responsiveness of PAR1 and PAR2, the cells were incubated with different concentrations of thrombin, tryptase, or trypsin, and the levels of secreted IL-6 were quantified by ELISA. As shown in Physique 2(a), incubation with thrombin (14C220 nM) resulted in a dose-dependent increase in IL-6 production. Incubation of HCECs with tryptase (55?220 nM) or trypsin (55?220 nM) also stimulated IL-6 production in a concentration-dependent manner (Figures 2(b) and 2(c)). Open in a separate window Open in a separate window Open in a separate window Physique 2 < .05 when compared to medium control. Thrombin cleavage of PAR1 exposes a new < .05 when compared to medium control. Effects of inhibiting protease activity and G-protein coupling on PAR-mediated IL-6 release The requirement for catalytic activity for thrombin and trypsin for stimulation of PAR1 and PAR2, respectively, was confirmed by incubating NBR13 thrombin with a selective inhibitor, hirudin, and trypsin with a selective inhibitor, soybean trypsin inhibitor. As depicted in Physique 4, the ability of thrombin and trypsin to induce IL-6 production by HCECs was completely abrogated when enzymes were treated with their specific inhibitors. In control studies, hirudin did not inhibit trypsin activity nor did soybean trypsin inhibitor inhibit thrombin activity on HCECs (data not shown). Open in a separate window Physique 4 < .05 when compared to medium control. It is well-recognized that this PAR-signaling pathway MK-6096 (Filorexant) requires G-protein coupling [5, 21]. Therefore, the effect of the G-protein-coupled receptor inhibitor, pertussis toxin, on thrombin- and tryptase-induced IL-6 production was tested. As shown in Physique 5, preincubation of HCECs with pertussis toxin for 30 minutes prior to the addition of thrombin or trypsin completely inhibited IL-6 release. Open in a separate window Physique 5 < .05 when compared.