Staphylococcus Aureus Peptidoglycan Upregulates Expression of Toll-Like Receptor 2, CD14, ICAM-1, HLA-DR and Release of TNF and IL-8 From Human Conjunctival Epithelial Cells

Abstract

Abstract: : Staphylococcal bacteria (S. aureus) are found in large colony counts on the skin of 90% of patients with atopic dermatitis and could play a role in the pathophysiology of atopic keratoconjunctivitis. S. aureus proteins can be recognized by Toll-like receptor 2 (TLR2), resulting in release of inflammatory cytokines. Purpose: To examine human conjunctival epithelial cells (EC) for expression of TLR2 and responses to S. aureus peptidoglycan (PGN). Methods: EC were enzymatically dispersed from multiple donor pools of cadaveric conjunctival tissues and cultured in 24 well plates on a fibronectin/collagen matrix. The resulting primary EC culture monolayers were incubated with and without S. aureus PGN and/or anti-TLR2 antibody for 24 hours. Supernates were harvested for cytokine (TNFα , IL-8) analysis by ELISA. EC were harvested with Trypsin-EDTA and double stained with anti-TLR2-APC (monoclonal biotinylated antibody followed by streptavidin-APC) versus anti-CD14-PE or anti-ICAM-1-PE versus anti-pan-HLA -FITC. Antibody staining was analyzed by flow cytometry. Results: PGN upregulated expression of all receptors examined, in a dose-dependent manner. PGN also stimulated a dose dependent increase in IL-8 release. Concentrations of IL-8 (expressed as ng/million cells minus unstimulated) went from constitutive levels (at 0.1 µ g/ml PGN) to 333.2 ± 52.2 and 1364.4 ± 66.4 (for 1.0 and 10 µ g/ml PGN respectively). A trend toward increased TNFα release with PGN was also observed. Anti-TLR2 stimulation alone had no effect on any parameters measured. Co-stimulation with anti-TLR2 and PGN further upregulated TLR2 staining over PGN alone but had no additional effect on any other parameters measured. Conclusions: Expression of TLR2 on conjunctival EC and activation by S. aureus PGN suggest that these cells may play an active immunological role in ocular defense against microbial pathogens.

Keywords: conjunctivitis • inflammation • Staphylococcus