IgE–Activated Human Conjunctival Mast Cell Supernates Upregulate TNFR –1 Expression on Conjunctival Epithelial Cells

Abstract

Abstract: : Conjunctival mast cells are an important source of TNFα in ocular surface inflammation. Our research further suggests that conjunctival mast cell mediators render conjunctival epithelial cells more sensitive to TNFα mediated ICAM–1 upregulation. Purpose: The purpose of this study was to examine whether TNFα mediated ICAM–1 upregulation by supernates from IgE–activated conjunctival mast cells is secondary to upregulation of the receptor for soluble TNFα, TNFR1. Methods: Human conjunctival mast cells and epithelial cells were enzymatically dispersed and purified from multiple donor pools of cadaveric conjunctival tissues. IgE–activated mast cell supernates were obtained from 90 min challenge of purified conjunctival mast cells with goat anti–human IgE. Conjunctival epithelial cells were incubated with and without IgE–activated mast cell supernates or pro–inflammatory cytokines (TNFα, IFNγ) for 24 hrs prior to harvesting with Trypsin–EDTA for analysis of ICAM–1 and TNFR1 receptor expression using two–color flow cytometry analysis. Results: IgE activated mast cell supernates increased both TNFRI and ICAM–1 expression. TNFR1 expression was detectable only on epithelial cells that also expressed high levels of ICAM–1. Conversely, ICAM–1 expression on cells expressing TNFR1 was 200–300 mean fluorescence intensity units greater than ICAM–1 expression on TNFR1 negative cells. Epithelial cells expressing high levels of TNFR1 responded to TNFα stimulation with greater upregulation of ICAM–1 compared to IFNγ stimulation, even though IFNγ was a more potent stimulator of ICAM–1 in the total population of epithelial cells. Conclusion: These results demonstrate that the mechanism of by which TNFα in IgE–activated conjunctival mast cell supernates upregulates ICAM–1 expression on conjunctival epithelial cells involves a coordinated upregulation of TNFR1 expression. Consequently, increased expression of TNFR1 on conjunctival epithelial cells may increase the potential for ICAM–1 upregulation in response to TNFα.

Keywords: conjunctiva • cytokines/chemokines • inflammation