Innate Immunity at the Ocular Surface: Spectrum of Antimicrobial Peptide Expression

Abstract

Abstract: : Purpose:We have shown that human ocular surface epithelia express a number of antimicrobial peptides: three ß–defensins (hBD1–3) and cathelicidin (LL–37). The purpose of this study was to explore if additional antimicrobial peptides are expressed by corneal and conjunctival epithelial cells and to study the antimicrobial activity of the peptides against common ocular pathogens in vitro. Methods: Expression of mRNA and protein of various antimicrobial peptides was determined by RT–PCR and immunostaining, respectively, in scraped human corneal epithelium, primary cultured human corneal epithelial cells (HCEC), conjunctival epithelial cells, and corneal tissue sections. In some experiments, HCEC were treated with 10ng/ml IL–1ß or TNF–α for 6 and 24 hours. Cells treated with serum–free culture media acted as controls. Antimicrobial assays were performed to assess peptide activity against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Staphylococcus epidermidis (SE). Results: All epithelial samples (n=3) constitutively expressed mRNA for two antimicrobial peptides: macrophage inflammatory protein (MIP–3α/CCL20) and thymosin ß–4 (Tß4). This expression was not modulated by IL–1ß or TNF–α. None of the epithelial samples expressed hBD–4, –5, –6, HE2ß1, histatins (Hist–1, –3), or liver–expressed antimicrobial peptides (LEAP–1, –2). Scraped corneal epithelium and cultured HCEC also expressed MIP–3α/CCL20 and Tß4 protein (n=2). Immunostaining showed the presence of MIP–3α/CCL20 and Tß4 throughout the entire epithelial layer (n=3). hBD–3 (EC₅₀= 0.9–5.3µg/ml) inhibited growth of all bacteria in a concentration dependent manner (n=3). hBD–2 (EC₅₀= 1.2+0.2µg/ml), MIP–3α/CCL20 and Tß4 (EC₅₀= 18.1+1.7µg/ml) were effective against PA, but were only weakly effective against staphylococcal strains (n=3). hBD–1 (EC₅₀=21.4+1.5µg/ml) showed activity against PA, but was not effective against SA or SE (n=3). Conclusions:Our data show that in addition to ß–defensins and cathelicidin, the ocular surface epithelia express MIP–3α/CCL20 and Tß4. The presence of various antimicrobial peptides at the ocular surface likely contributes to the innate response protecting the eye against infection.

Keywords: cornea: epithelium • cornea: basic science • Pseudomonas