Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay

• Published in: Cornea Vol. 29; no. 9; p. 1048
• Main Authors: Zheng, Xiaofen, De Paiva, Cintia S, Rao, Kavita, Li, De-Quan, Farley, William J, Stern, Michael, Pflugfelder, Stephen C
• Format: Journal Article
• Language: English
• Published: United States 01.09.2010
• Subjects: Adult; Aged; Animals; Biological Assay - methods; Bromodeoxyuridine; Cell Division; Cell Line; Cell Proliferation; DNA - biosynthesis; Dry Eye Syndromes - metabolism; Enzyme-Linked Immunosorbent Assay; Epithelial Cells - metabolism; Humans; Lung - cytology; Middle Aged; Tears - metabolism; Transforming Growth Factor beta1 - metabolism; Transforming Growth Factor beta2 - metabolism
• ISSN: 1536-4798
• DOI: 10.1097/ICO.0b013e3181cf98ff

To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta 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-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 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-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta 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-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.