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A.M. McDermott, R.L. Redfern, S. Narayanan, R.J. Proske; Effect of Defensins on Human Corneal Epithelial Cell Migration . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4754.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose : Defensins are antimicrobial peptides expressed at the ocular surface and present in the tear film. In some cell types defensins have been shown to modulate behaviours such as migration, proliferation and cytokine secretion. Here we have investigated the ability of defensins to modulate migration of human corneal epithelial cells (HCEC). Methods : Experiments were carried out using SV40-transformed HCEC.Blindwell migration assay using 10µm filters: 105 HCEC in serum-free culture media were placed in the upper chambers. Human neutrophil peptide-1 (HNP-1) or human ß-defensin-2 (hBD-2) diluted in serum-free culture media were added (concentration range 0.125 to 1 µM) to, either the upper chamber, bottom chamber or both. Culture media containing 10% serum added to the bottom chamber was used as a positive control. After 16hrs of incubation at 37oC, the filters were stained and number of migrated cells counted. In some experiments HCEC were pretreated with IL-1ß (10ng/ml) for 24 hours. Wounding assay : HCEC were grown to confluency and treated with mitomycin C (50µg/ml, 30 min) to prevent proliferation. Then, a circular wound (3.5mm diameter) was created using a probe chilled in liquid nitrogen. The cells were treated with hBD-2 (0.1 to 500nM) for 24 hours at which time the cells were fixed, stained and the area of the wound calculated using NIH image software. Results : HNP-1 and hBD-2 did not stimulate HCEC chemotaxis or chemokinesis (n= 1-5). Pre-treatment of HCEC with IL-1ß, which upregulates CCR6, a putative receptor mediating chemotactic effects of hBD-2 in other cell types, did not induce chemotaxis in response to hBD-2 (n=2). The percent wound closure achieved by 24 hrs was 22-30% in hBD-2 treated cells and 33% in control untreated cells (n=2). Conclusions : Defensins do not significantly modulate HCEC migration in vitro. Thus, defensins may be valuable as a topical treatment for preventing infection after corneal injury that will not impair wound closure.
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