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J. L. Stahl, E. B. Cook, F. M. Graziano, N. P. Barney; Regulation of Expression and Shedding of the Soluble Receptor for TNFa, TNFR1, in Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5174.
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© ARVO (1962-2015); The Authors (2016-present)
Upregulation of ICAM-1 expression on the ocular surface is an important feature of ocular allergic inflammation. Previous in vitro studies have demonstrated that mast cell-derived TNFα stimulation is critical to upregulation of ICAM-1 on conjunctival epithelial cells. Shedding of TNFR1 by TNFα Converting Enzyme (TACE) is believed to be a primary mechanism for regulation of soluble TNFα-mediated events, yet this process has not been examined in human conjunctival epithelial cells. The purpose of this study was to examine regulation of TNFR1 expression and shedding by the zinc-dependent enzyme, TACE, on conjunctival epithelial cells.
Primary human conjunctival epithelial cells were enzymatically dispersed and purified from multiple donor pools of cadaveric conjunctival tissues. Conjunctival epithelial cells (primary and IOBA-NHC) were incubated with and without PMA or TNFα for 24 hrs. In some cases, conjunctival epithelial cells were pre-treated with either TAPI2 (TACE-specific inhibitor), EDTA, or ZnCl. Supernates were collected for analysis of soluble TNFR1 (sTNFR1) via ELISA and cells were harvested for analysis of ICAM-1 and TNFR1 receptor expression using two-color flow cytometry analysis.
In both primary and IOBA-NHC human conjunctival epithelial cells TIMP2 inhibited release of sTNFR1 receptor and enhanced surface expression of TNFR1 in a dose-dependent manner. Increased TNFR1 expression correlated with increased sensitivity to TNFα-mediated upregulation of ICAM-1. Incubation with EDTA and ZnCl also inhibited shedding of sTNFR1 in a dose dependent manner.
Inhibition of TNFR1 shedding by the mechanisms examined confirms that this process is TACE dependent and that TACE may have an inhibitory zinc-binding site. Since these results suggest that the level of TNFR1 expression on conjunctival epithelial cells is an important factor in regulation TNFα-mediated ICAM-1 upregulation, the mechanisms examined may provide significant targets for pharmaceutical intervention in ocular inflammation.
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