June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Expression of PAR1 and 2 on Human Corneal Epithelial Cells and Induction of Pro-inflammatory Cytokine Secretion by Acanthamoeba MIP-133 and aPA
Author Affiliations & Notes
  • Hassan Alizadeh
    Cell Biology and Anatomy, UNTHSC Fort Worth TX, Fort Worth, TX
    North Texas Eye Research Institute, UNTHSC, Fort Worth, TX
  • Mahshid Abdi
    Cell Biology and Anatomy, UNTHSC Fort Worth TX, Fort Worth, TX
    North Texas Eye Research Institute, UNTHSC, Fort Worth, TX
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5995. doi:
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      Hassan Alizadeh, Mahshid Abdi; Expression of PAR1 and 2 on Human Corneal Epithelial Cells and Induction of Pro-inflammatory Cytokine Secretion by Acanthamoeba MIP-133 and aPA. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5995.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Protease activated receptors (PARs) are G-protein coupled receptors, which initiate inflammatory responses when activated by serine proteinase. We hypothesized that MIP-133 (mannose induced protein) and aPA (Acanthamoeba plasminogen activator) activate PARs on the corneal epithelial cells, resulting in signal transduction and production of pro-inflammatory cytokines that modulate corneal inflammation in Acanthamoeba keratitis.

Methods: Human corneal epithelial cells were incubated with or without purified 15 ug aPA or 7.5 ug MIP-133 for 24 hr. As a positive control the cells were incubated with a 10 uM-human thrombin, PAR1 agonist. Corneal epithelial cells were also stimulated with a PAR2 agonist such as 10nM bovine trypsin, and 100 uM peptide corresponding to the receptor-activating tethered domains of PAR2 (SLIGRL-NH2). The expression of PAR1 and PAR 2 mRNA in human corneal epithelial cells was examined by RT-PCR. IL-6 and IL-8 gene expression was determined by RT-PCR and the levels of secreted IL-8 were determined by ELISA.

Results: Stimulation of corneal epithelial cells with a PAR1 agonist (thrombin) and aPA or PAR2 agonist (SLIGRL-NH2), trypsin and aPA resulted in upregulation of PAR1 and PAR2 mRNA and a significant IL-8 (P<0.05) protein production (10-13 fold). Thrombin failed to upregulate IL-6 and IL-8 genes expression in HEC cells, however, MIP-133 induced upregulation of IL-8 mRNA expression. Thrombin and MIP-133 were able to induce IL-8 protein production by corneal epithelial cells (2 fold).

Conclusions: MIP-133 and aPA interact with PAR1 and PAR2 on HCE cells and activate pro-inflammatory cytokines/chemokines from HCE cells. Disruption of PAR1 and PAR2 activity might have a major impact on preventing inflammatory responses in Acanthamoeba keratitis and bacterial infection.

Keywords: 402 Acanthamoeba • 480 cornea: basic science • 594 microbial pathogenesis: experimental studies  
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