Characterization and Functionality of CXCR4 Chemokine Receptor and SDF-1 in Human Corneal Fibroblasts

Abstract

Abstract: : Purpose: The aim of the study was to investigate whether cultured human corneal fibroblasts express functional chemokine CXCR4 receptors on their cell surface and to determine the presence of its specific ligand, SDF-1a (CXCL12), in corneal fibroblasts. Methods: Human corneal fibroblast cultures were obtained using human donor corneas. CXCR4 receptors were characterized using binding studies and autoradiography with [125I]SDF-1a. Functionality of CXCR4 receptors was assessed by intracellular calcium measurement using a dynamic imaging microscopy system. CXCR4 and SDF-1a mRNA were detected in human corneal fibroblasts by means of the reverse transcriptase polymerase chain reaction (RT-PCR). The CXCR4 protein was detected by Western-blot analysis. Results: [125I]SDF-1a specifically bound to cultured corneal fibroblasts with a KD value of 8.3 ± 1.2 nM. The presence of CXCR4 was confirmed by autoradiography of the radioligand on slices of corneal stroma. SDF-1a induced a rapid and transient intracellular calcium increase in cultured corneal fibroblasts that was blocked by the specific antagonist bicyclam. Moreover, a 48 kDa-protein was detected by Western-blot analysis of corneal fibroblast extracts, using a specific CXCR4 polyclonal antibody. Reverse transcriptase polymerase chain reaction showed the expressions of both CXCR4 and SDF-1a mRNAs in human corneal fibroblasts. Conclusions: These results indicate for the first time that cultured human corneal fibroblasts express the chemokine receptors CXCR4 and its ligand SDF-1a. This latter might exert physiological effects on cornea and could be involved in pathological conditions such as corneal angiogenesis.

Keywords: cornea: stroma and keratocytes • cytokines/chemokines • receptors: pharmacology/physiology