In vivo Expression of Toll–Like Receptor–2 on Conjunctival Epithelial Cells Is Increased in Atopic Keratoconjunctivitis

Abstract

Abstract: : Purpose: Increased colonization with Staphylococcus aureus (SA) may play a pro–inflammatory role in atopic keratoconjunctivitis (AKC). We previously demonstrated that human conjunctival epithelial cells are activated by a commercially prepared extract of SA cell wall via interaction with Toll–like receptor 2 (TLR2) expressed on human conjunctival epithelial cells in vitro. The purpose of this study was to examine human conjunctival epithelial cells derived from subjects with AKC, seasonal allergic conjunctivitis (SAC), and non–allergic subjects for expression of TLR2. Methods: For in vivo studies, conjunctival epithelial cells were acquired via impression cytology, fixed, and examined for TLR2 expression via flow cytometry. Anti–CD45 was used to gate out contaminating leukocytes. For in vitro studies, human conjunctival epithelial cells were enzymatically dispersed and purified from multiple donor pools of cadaveric conjunctival tissues and cultured on 24–well plates. Conjunctival mast cells were challenged with anti–IgE antibody to obtain activated mast cell supernates. Conjunctival epithelial cells were incubated with the activated mast cell supernates for 24 hours, then harvested using trypsin–EDTA for immunostaining via flow cytometry. Results: Staining of conjunctival epithelial cells for TLR2 was positive in 4/5 subjects with AKC, 3/5 subjects with SAC, and 0/3 non–allergic subjects. Intensity of TLR2 staining was significantly greater on conjunctival epithelial cells obtained from AKC subjects compared to conjunctival epithelial cells obtained from non–allergic subjects (p < 0.05). These results suggest that mast cell activation promotes TLR2 expression by conjunctival epithelial cells. In vitro examination of cultured human conjunctival epithelial cells following incubation with IgE–activated conjunctival mast cell supernates demonstrated upregulation of TLR2 expression. Conclusions: While TLR2 may not be constitutively expressed on conjunctival epithelium, ocular allergic inflammation appears to increase TLR2 expression, which is significant in AKC. This increased expression could promote ocular defense against colonizing pathogenic Gram–positive bacteria.

Keywords: inflammation • conjunctivitis • microbial pathogenesis: experimental studies