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Yuichi Uchino, Pablo Argueso; Galectin-3 Mediates IL1β Responses in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5650.
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© ARVO (1962-2015); The Authors (2016-present)
Galectin-3, a multimeric carbohydrate-binding protein expressed by the ocular surface epithelia, is a pleiotropic molecule with immunomodulatory functions. Here, we investigated whether galectin-3 modulates IL1β responses by evaluating IL-8 secretion in human corneal epithelial cells.
The presence of IL-1 receptor 1 (IL1R1) and IL-1 receptor accessory protein (IL1RAcP) was evaluated by immunofluorescence in healthy human corneal epithelial tissue. Small interfering RNA (siRNA) targeting galectin-3 was introduced into stratified cultures of human corneal-limbal epithelial (HCLE) cells by lipofectamine-mediated transfection. Stratified HCLE cell cultures were treated for 6 hrs with serum-free growth-media alone or with recombinant human IL-1β (10 ng/ml). The presence of IL-8 in cell culture supernatants and IL1R1 in whole cell lysates was quantified by Western blot. The cell surface localization and interaction between IL1R1 and galectin-3 were determined by biotinylation and galectin-3 affinity chromatography, respectively.
IL1R1 and IL1RAcP were detected throughout the entire epithelia in human corneal tissue. Treatment of stratified HCLE cells with galectin-3 siRNA decreased galectin-3 protein biosynthesis by 85%. Secretion of IL-8 following IL-1β treatment was impaired in galectin-3 knockdown cells compared to scramble control cells. In these experiments, the amount of IL1R1 in the cell lysates was not affected by either IL-1β stimulation or galectin-3 knockdown. Similarly, IL-1β treatment did not alter the amount of IL1R1 on apical membranes. In addition, galectin-3 failed to bind IL1R1 by affinity chromatography.
Galectin-3 regulates IL-1-mediated inflammatory responses in human corneal epithelial cells. However, the molecular basis for this regulatory mechanism remains to be elucidated.
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