Stimulation of interleukin-6 production by human conjunctival epithelial cells (HCEC) with thrombin and tryptase

Abstract

Rationale Conjunctival epithelial cells protect ocular tissues and participate in the regulation of allergic inflammation by releasing numerous cytokines such as IL-6, IL-8, eotaxin, RANTES, and TNF-α. Serine proteases, acting through a G-protein coupled family of receptors known as protease activated-receptors (PARs), also participate in the inflammatory response. Thrombin and tryptase are known to activate many cell types via PAR-1 and PAR-2, respectively. In this study, we examined the effects of thrombin and tryptase on HCEC activation and demonstrated the presence of PAR-1 by RT-PCR and both PAR-1 and PAR-2 by immunoflourescence. Methods Confluent HCECs (Clone 1-5c-4, ATCC) were cultured with thrombin (1-25 U/ml), tryptase (1-50 mU/ml), or TNF-α (100-500 U/ml) for 24 hours. The IL-6 levels in culture supernatants were assayed by ELISA. The constitutive expression of PAR mRNA and proteins were determined by RT-PCR and immunoflourescence, respectively. Results Incubation of HCECs with thrombin resulted in a dose dependent statistically significant increase in IL-6 production (p<.01) compared to medium treated cells. Thrombin-induced IL-6 production was inhibited by the thrombin inhibitor, hirudin, and by the G-protein coupling inhibitor, pertussis toxin. Tryptase also stimulated IL-6 production in HCECs, but the effect was of lower in magnitude when compared to that of thrombin. HCECs express PAR-1 mRNA. PAR-1 and PAR-2 were also evident by immunoflourescence. Conclusions The results demonstrate that HCECs express PAR-1 and PAR-2 and produce IL-6 in response to both thrombin and tryptase. The response to thrombin, a PAR-1 agonist, was of higher magnitude than tryptase, a PAR-2 agonist.