Abstract
Protease-activated receptors (PARs) are G-protein-coupled receptors which initiate inflammatory responses when activated by specific serine proteases. This study was conducted to examine whether human conjunctival epithelial cells (HCECs) express functionally active PAR1 and PAR2 using Chang conjunctival epithelial cells as in vitro model. We performed RT-PCR and immunofluorescence analyses to determine the expression of PAR1 and PAR2, and monitored the production of IL-6 after activating HCECs with PAR1 activating agents (thrombin or TFLLRN) or PAR2 activating agents (tryptase, trypsin, or SLIGKV). The results show that HCECs constitutively express PAR1 and PAR2 mRNA and proteins, and produce significant amounts of IL-6 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.
Highlights
Allergic inflammation of the ocular conjunctiva is associated with increased mast cell mediators in tear fluid, and the recruitment of activated eosinophils, mast cells, and lymphocytes [1]
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
The results of the present study demonstrate for the first time that human conjunctival epithelial cells (HCECs) constitutively express PAR1 and PAR2 mRNA and protein (Figures 1(a) and 1(b))
Summary
Allergic inflammation of the ocular conjunctiva is associated with increased mast cell mediators in tear fluid, and the recruitment of activated eosinophils, mast cells, and lymphocytes [1]. Ocular epithelial cells are active participants in the regulation of allergic inflammation via expression of adhesion molecules and elaboration of proinflammatory cytokines and chemokines [2]. Recent work has highlighted the potential role of protease-activated receptors (PARs) in stimulating cytokine production by respiratory epithelium [3]. 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 N-terminal domains. Four subtypes of PARs have been described [6,7,8,9], and among these, PAR1 and PAR2 are widely expressed in many cell types including endothelial cells, platelets, bronchial epithelial cells, fibroblasts, mast cells, neurons, leukocytes, eosinophils, airway and vascular smooth muscle cells, keratinocytes, and renal tubular cells [3,4,5,6,7,8,9,10,11,12,13,14]
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