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K.G. J. Ooi, G. Galatowicz, V.L. Calder, H.M. A. Towler, S.L. Lightman; Cytometric Bead Array for the Detection of 6 Cytokines in Aqueous Humor of Uveitis Patients . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2813.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine levels of IL–2, IL–4, IL–5, IL–10, TNFα and IFN–γ in aqueous humor (AH) taken from patients with active panuveitis, anterior uveitis (AU) or controls using cytometric bead array (CBA) and compare with IL–4, IL–10, TNFα and IFN–γ as detected by ELISA. Methods: Control AH was obtained from 10 patients undergoing phacoemulsification while AH from 36 active uveitis patients was collected via anterior chamber paracentesis. Samples were centrifuged 400 x g for 5 minutes and supernatants stored frozen (–70 degrees C) until testing. 10 ml samples (standards or test) were added to 50 ml of a cocktail of capture beads and detector antibodies.The mixture was incubated 18hr at room temperature in the dark. Excess unbound detector antibody was washed off before two colour flow cytometric analysis and comparison with ELISA cytokine detection. Results: ELISA was comparable to CBA in the level of detection of IL–10 and IFN–γ but CBA proved to be more sensitive than ELISA in the detection of IL–4 and TNFα. Levels of IL–2, IL–4 and TNFα were not detected to any level of significance in the uveitis groups over controls. Significantly increased levels of IFN–γ were detected in both AU (10–10000 pg/ml) and panuveitis (50–1000 pg/ml) patients (p<0.01). Significantly decreased levels of IL–5 were detected in the panuveitis group as compared to controls (p<0.01) and the AU group (p<0.05). The increased levels of IFN–γ correlated with decreased levels of IL–4 and IL–5 within individual AH samples. Significantly raised levels of IL–10 were detected in the panuveitis group on steroids as compared to controls (p<0.01). Conclusions: CBA allows multiple cytokine analytes to be more rapidly quantified in a small, single, AH volume, with better reproducibility and sensitivity than ELISA. The elevated IFN–γ suggests Th1 polarization in active uveitis while the decreased IL–5 production might suggest that Th1 polarity is more marked with greater uveal tract involvement in panuveitis. The relative elevation of Th1 versus Th2 cytokines within individual AH samples also illustrates another advantage of CBA in its ability to compare relative cytokine levels within single samples. The significantly raised IL–10 levels in the steroid treated group suggest a glucocorticoid up–regulation of IL–10.
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