The cellular basis for biocide-induced fluorescein hyperfluorescence in mammalian cell culture.
Clinical examination of the ocular surface is commonly carried out after application of sodium fluorescein in both veterinary and medical practice by assessing the resulting 'staining'. Although localized intensely stained regions of the cornea frequently occur after exposure to 'adverse' clinical stimuli, the cell biology underlying this staining is unknown, including whether intense fluorescein staining indicates the presence of damaged cells. Ocular exposure to certain contact lens multipurpose solutions (MPS) gives rise to intense fluorescein staining referred to as solution induced corneal staining (SICS), and we have made use of this phenomenon with Vero and L929 cell culture models to investigate the fundamental biology of fluorescein interactions with cells. We found that all cells take up fluorescein, however a sub-population internalize much higher levels, giving rise to brightly staining 'hyperfluorescent' cells within the treated cultures, which contain fluorescein throughout the cell cytoplasm and nucleus. The numbers of these hyperfluorescent cells are significantly increased after exposure to MPS associated with SICS. Surprisingly, hyperfluorescent cells did not show higher levels of staining with propidium iodide, a marker of lysed cells. Consistently, treatment with the cytolytic toxin benzalkonium chloride resulted in almost all cells staining with propidium iodide, and the complete abolition of fluorescein hyperfluorescence. Finally we found that internalization of fluorescein and its loss from treated cells both require cellular activity, as both processes were halted after incubation at 4 °C. We conclude that fluorescein hyperfluorescence can be replicated in three diverse cell cultures, and is increased by MPS-treatment, as occurs clinically. The process involves the concentration of fluorescein by a sub-population of cells that are active, and does not occur in lysed cells. Our data suggest that corneal staining in the clinic reflects active living cells, and is not directly caused by dead cells being produced in response to adverse clinical stimuli.
Eurolens Research, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom ; Medical Device Biology Group, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
May M
MM
Bakkar
Medical Device Biology Group, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
Luke
L
Hardaker
Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
Peter
P
March
Eurolens Research, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom ; Medical Device Biology Group, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
Philip B
PB
Morgan
Eurolens Research, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom ; Medical Device Biology Group, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
Carole
C
Maldonado-Codina
Medical Device Biology Group, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
Curtis B
CB
Dobson
D000818:Animals; D000891:Anti-Infective Agents, Local / Q000494:pharmacology*; D001548:Benzalkonium Compounds / Q000494:pharmacology; D002460:Cell Line; D002522:Chlorocebus aethiops; D004347:Drug Interactions; D019793:Fluorescein / Q000494:pharmacology*; D051379:Mice; D011419:Propidium; D012996:Solutions; D014709:Vero Cells
D000891:Anti-Infective Agents, Local; D001548:Benzalkonium Compounds; D012996:Solutions; D011419:Propidium; D019793:Fluorescein