Evaluation of the Transforming Growth Factor-β Activity in Normal and Dry Eye Human Tears by CCL-185 Cell Bioassay


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Abstract

Purpose:To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-β) in human tear fluid from normal subjects and patients with dry eye.Methods:Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-β by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-β on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-β concentration by Luminex microbead assay, and TGF-β activity by the CCL-185 cell growth inhibition bioassay.Results:The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-β1 concentration (R2 = 0.919) and confirmed by BrdU assay (R2 = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-β1 concentration in the CCL-64 line (R2 = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-β1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-β1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-β1 concentrations. Levels of immuodetectable TGF-β1 and TGF-β2 were similar in normal and DE tears. TGF-β bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 ± 10481.9 pg/mL) than those in normal controls (4129.3 ± 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-β bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-β in DE tears and 37.6% TGF-β in normal tears were found to be biologically active.Conclusions:The CCL-185 cell assay was found to be a suitable tool for assessing TGF-β activity in human tears. Tear TGF-β bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-β1 transcripts in the conjunctival epithelium have been previously detected.

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