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C. Baudouin, L. Bensoussan, C. Blondin, C. Creuzot-Garcher, P. Hamard, J. Warnet, F. Brignole; IL-6 and IL-8 Assessment in Ocular Surface Inflammation using Flow Cytometry . Invest. Ophthalmol. Vis. Sci. 2003;44(13):677.
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Purpose: To analyze by flow cytometry the expression of inflammatory markers HLA-DR, IL-6 and IL-8 in conjunctival epithelial cells obtained by impression cytology (IC). Methods: 83 impression cytology samples were obtained from 60 patients, 49 patients suffering from chronic ocular inflammatory disorders and 15 subjects without any ophthalmologic disease. Immunofluorescence techniques were performed using monoclonal antibodies against HLA-DR antigen (Dako), IL-6 and IL-8 (BD Biosciences). Both membrane (m) and cytoplasmic (c) expressions were assessed on a Coulter XL Epics flow cytometer, as fluorescence levels and percentages of positive cells. Fluorescence levels were quantified as arbitrary units of fluorescence (AUF), using fluorescent calibrated beads (QIFIKIT for HLA-DR and Dako-Fluorospheres for IL-6 and IL-8). Results: A significant increase of inflammatory marker expressions (percentages and AUF) was observed for HLA-DR (respectively p=0.004; p=0.007), cIL-6 (p<0.0001; p=0.003) and cIL-8 (p<0.0001; p=0.001) in conjunctival cells from inflammatory patients when compared to controls. Percentages and AUF of mIL-6 were higher in the control group than in the inflammatory one (respectively p=0.001; p<0.0001), whereas percentage of mIL-8 but not AUF was found significantly increased in the inflammatory group (p=0.04) as compared to controls. A significant positive correlation was found between the expression of HLA-DR and cIL-6 and cIL-8 (respectively p=0.0004; p=0.0005) as well as between cIL-6 and cIL-8(p<0.0001). Conclusions: Flow cytometry is a new reliable standardized technique allowing objective quantification of inflammatory processes in the conjunctiva. We showed the increase of HLA-DR, IL-6 and IL-8 expressions by epithelial cells in chronic inflammation of the ocular surface. Therefore, this method may represent a valuable tool for exploring the cytokine participation in inflammatory and allergic ocular surface disorders.
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