Novel Role for Thy–1 in Mediating Corneal Fibroblast Attachment to Extracellular Matrix Proteins

Abstract

Purpose: : We have previously shown that Thy–1, a known cell–cell adhesion protein, is expressed by corneal fibroblasts and myofibroblasts. Here we have investigated the role of Thy–1 in mediating attachment of corneal fibroblasts to neutrophils and to extracellular matrix proteins.

Methods: : Human corneal keratocytes were isolated by collagenase digestion then cultured in 10% serum. To study cell–cell interaction, freshly isolated human neutrophils were labeled with the fluorescent dye Calcein–AM then incubated with confluent cultures of corneal fibroblasts for 1 hr. The fluorescence due to the attached neutrophils was then quantitated in a plate reader. To study cell–matrix interaction, corneal fibroblasts pre–loaded with Calcein–AM were incubated with plates coated with collagen I, fibronectin or collagen III for 30 min. The fluorescence due to the attached fibroblasts was then quantitated in a plate reader. The involvement of Thy–1 was established by comparing cell–cell/matrix adhesion in untreated corneal fibroblasts to those treated with phospholipase C (PLC, cleaves GPI linked proteins such as Thy–1 from the cell surface) or an anti–Thy–1 antibody.

Results: : Neutrophils were found to attach to corneal fibroblasts but neither pre–treatment with PLC (n=7) nor an anti–Thy–1 antibody (n=3) interfered with this interaction between the two cell types. Corneal fibroblasts attached to collagen I, fibronectin and collagen III. PLC treatment reduced fibroblast attachment to fibronectin (21%, n=6) and collagen III (40%, n=3), but not collagen I. Anti–thy–1 antibody reduced fibroblast attachment to collagen III (50%, n=7).

Conclusions: : Thy–1 does not mediate the interaction between neutrophils and corneal fibroblasts, but is able to partially mediate an interaction between fibroblasts and collagen III. The latter, like corneal fibroblasts, is present after corneal stromal injury, thus Thy–1 may function to help sequester fibroblasts to a site of injury.

Keywords: cornea: stroma and keratocytes • cornea: basic science • wound healing