Expression of Trypsin and Modulation of PAR-2, a Trypsin Receptor, in Human Conjunctival Epithelial Cells

Abstract

Purpose:: Allergic conjunctivitis (AC) is one of the most commonly encountered ocular disorders. The pathogenesis of allergic conjunctivitis involves binding of environmental allergens to mast cell membrane IgE, causing cell degranulation and the release of various inflammatory mediators including histamine and tryptase. Severe and chronic cases of AC can lead to remodeling of the ocular surface. Current therapies are often unsatisfactory. Proteinase Activated Receptor 2 (PAR-2) is a potent mediator of tissue inflammation and known to be activated by mast cell tryptase and cellular derived trypsin in a number of inflammatory disorders. We have previously reported that human conjunctival epithelial cells (CE) express functional PAR-2 that can be activated by mast cell tryptase to initiate inflammatory responses. Because trypsin is also a potent PAR-2 agonist, we investigated if CE could be a source of this enzyme and if it could contribute to the pathogenesis of allergic conjunctivitis. We propose that mast cell tryptase, together with CE derived trypsin, play key roles in the pathogenesis of AC.

Methods:: The expression of trypsin in human CE and conjunctival tissue was determined by RT-PCR and immunohistochemistry. Trypsin and PAR-2 modulation studies were performed using quantitative RT-PCR.

Results:: Our results indicated that human CE as well as freshly isolated human conjunctival tissue constitutively express trypsin mRNA and protein. Trypsin mRNA expression in CE was increased by the inflammatory cytokines IL-1a and TNFa. We have shown that in CE, trypsin induces a rapid increase in the intracellular calcium concentration, NF-kB nuclear translocation, increase in IL-1a, IL-1b, IL-6, IL-8, and TNFa mRNA expression, and IL-6 and IL-8 protein secretion in a time and dose response manner. We now further demonstrate the PAR-2 specificity of these responses by the blocking effect of anti-PAR2 antibodies in selected studies. We have also investigated the factors that modulate PAR-2 expression in human CE. Our results indicate that the cytokines IL-1a, IL-6 and IL-4, but not TNFa, were capable of inducing an increase in PAR-2 mRNA expression. Mast cell tryptase also caused PAR-2 upregulation, but interestingly, trypsin did not.

Conclusions:: These studies indicate for the first time that human CE express the protease trypsin that can induce PAR-2 activation and initiate inflammatory activities proposed to mediate the pathophysiological changes observed in AC. Modulation of epithelial PAR-2 in conjunctiva may result in a novel target for treating this common inflammatory ocular disorder.

Keywords: conjunctivitis • receptors • cytokines/chemokines