Abstract
Abstract: :
Purpose: We have previously shown that Thy–1, a cell surface protein, can be used to distinguish corneal fibroblasts and myofibroblasts from normal keratocytes (Pei, Y et al. IOVS; 44:ARVO E–abstract # 869). In this study we further characterized Thy–1 expression by human corneal fibroblasts and investigated the potential role of Thy–1 as an adhesion molecule mediating attachment of monocytes and corneal fibroblasts. Methods: Human corneal keratocytes were isolated by collagenase digestion then cultured in 10% fetal bovine serum (FBS) or 10% FBS and transforming growth factor–ß (5ng/ml) to obtain corneal fibroblasts and myofibroblasts respectively. Thy–1 mRNA and protein expression by different passages (0 to 4) of corneal fibroblasts were assessed by RT–PCR and western blotting. Immunolabeling for Thy–1 was performed on passage 0 fibroblasts 1, 2, 4 and 6 days after seeding the cells. The ability of the monocyte cell line U937 to attach to cultured fibroblasts and myofibroblasts was assessed in an adhesion assay using U937 cells pre–loaded with Calcein–AM. The effects of pre–treating fibroblasts with Thy–1 antibody (1– 50 µg/ml, 10 min – 3 hrs), intercellular adhesion molecule (ICAM) –1 antibody (1–25 µg/ml, 1 hr) and glycosylphosphatidylinositol (GPI) specific phospholipase C (100– 500 mU, 1 hr) on cell attachment were also investigated. Results: Strong Thy–1 mRNA and protein expression was present in all passages of corneal fibroblasts (n=2). In passage 0, positive Thy–1 labeling was first detected 6 days after seeding the cells (n=2). U937 monocytes attached to both corneal fibroblasts and myofibroblasts, approximately 25% of the added cells attached over 30 minutes (n=2). Thy–1 antibody or GPI specific phospholipase C did not significantly inhibit monocyte attachment to corneal fibroblasts, while ICAM–1 antibody caused a 40% inhibition (P<0.05, n=4). Conclusions: The induction of Thy–1 expression is consistent with the transformation of keratocytes to the corneal fibroblast phenotype. The results suggest that Thy–1 is not directly involved in mediating monocyte adhesion to keratocyte repair phenotypes. Thy–1 may mediate interaction with other cell types such as neutrophils or may have a role unrelated to cell–cell adhesion during wound healing.
Keywords: cornea: stroma and keratocytes • wound healing • cell adhesions/cell junctions