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EB Cook, JL Stahl, FM Graziano, NP Barney; Expression of Toll-Like Receptors 2 and 4 on Human Conjunctival Epithelial Cells and Mast Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1591.
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Purpose: Staphylococcal bacteria (S. aureus) are found in large colony counts on the skin of 90% of patients with atopic dermititis and could play a role in the pathophysiology of atopic keratoconjunctivitis through several mechanisms. One of these mechanisms involves recognition of S. aureus proteins by a Toll-like receptor (TLR), specifically TLR2, resulting in release of inflammatory cytokines such as TNF<font face="Symbol"≷a</font≷. The purpose of this study was to examine conjunctival epithelial and mast cells, both of which are involved in ocular inflammation, for expression of two TLRs, 2 and 4. Methods: Conjunctival epithelial cells and mast cells were acquired by enzymatic digestion of cadaveric conjunctival tissues and Percoll gradient separation. Epithelial cell monolayers were cultured to confluency (≷99% purity by cytokeratin staining) and pre-incubated with and without cytokines (IFN<font face="Symbol"≷g</font≷, TNF<font face="Symbol"≷a</font≷) for 24 hrs prior to immunostaining. Mast cells were purified by a second Percoll gradient separation (≷ 90% purity by Wright’s Stain). The cells were immunostained with biotinylated monoclonal antibodies to TLR2 and 4, followed by secondary affinity binding with allophyocyanin (APC) labeled streptavidin and analysis using flow cytometry. Mast cells were double stained with a phycoerythrin (PE) labeled anti-Kit monoclonal antibody to facilitate analysis of mast cells only. CD14 (co-receptor for TLRs) staining was also measured on conjunctival epithelial cells using a monoclonal anti-CD14 PE antibody. Results:Unstimulated epithelial cells stained positive for TLR2 (5.34±2.11%). Pre-incubation with IFN<font face="Symbol"≷g</font≷ increased TLR2 staining on conjunctival epithelial cells (10.72±5.39%). Staining for TLR4 was negligible on unstimulated epithelial cells (1.22±0.54%) and was marginally upregulated with both IFN<font face="Symbol"≷g</font≷and TNF<font face="Symbol"≷a</font≷(2.44±0.89% and 2.19%1.7% respectively). Positive staining for TLR2, could also be detected on conjunctival mast cells (16%), but not TLR4. CD14 expression was minimal on conjunctival epithelial cells (2.39±1.02%) but was increased by pre-incubation with IFN<font face="Symbol"≷g</font≷and TNF<font face="Symbol"≷a</font≷ (3.53±0.06% and 4.34±0.71% respectively). Conclusion: Expression of TLR2 on conjunctival epithelial cells and mast cells suggests that these cells play an active role in ocular defense against microbial pathogens.
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