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Stimulation of interleukin-6 production by human conjunctival epithelial cells (HCEC) with thrombin and tryptase
Abstracts S177
SUNDAY
J ALLERGY CLIN IMMUNOL VOLUME 113, NUMBER 2
609
Stimulation of Interleukin-6 Production by Human Conjunctival Epithelial Cells (HCEC) With Thrombin and Tryptase
T. J. Nickel, M. H. Kabir, J. Talreja, K. Dileepan, D. J. Stechschulte; Allergy, Immunology, and Rheumatology, University of Kansas, Kansas City, KS. 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. Funding: Carey Arthritis Fund
SUNDAY
J ALLERGY CLIN IMMUNOL VOLUME 113, NUMBER 2
609
Stimulation of Interleukin-6 Production by Human Conjunctival Epithelial Cells (HCEC) With Thrombin and Tryptase
T. J. Nickel, M. H. Kabir, J. Talreja, K. Dileepan, D. J. Stechschulte; Allergy, Immunology, and Rheumatology, University of Kansas, Kansas City, KS. 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. Funding: Carey Arthritis Fund