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


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-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.


RESULTS
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.


CONCLUSIONS
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.
Zheng
Xiaofen
X
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX 77030, USA.
De Paiva
Cintia S
CS
Rao
Kavita
K
Li
De-Quan
DQ
Farley
William J
WJ
Stern
Michael
M
Pflugfelder
Stephen C
SC
D000328:Adult; D000368:Aged; D000818:Animals; D001681:Biological Assay / Q000379:methods; D001973:Bromodeoxyuridine; D002455:Cell Division; D002460:Cell Line; D049109:Cell Proliferation; D004247:DNA / Q000096:biosynthesis; D015352:Dry Eye Syndromes / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology; D008875:Middle Aged; D013666:Tears / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D053781:Transforming Growth Factor beta2 / Q000378:metabolism*
D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D004247:DNA; D001973:Bromodeoxyuridine