To study the mechanism of collagen degradation by keratocytes, we deve
loped the new in vitro model in which keratocytes were cultured three-
dimensionally in a collagen matrix. Subcultured rabbit keratocytes wer
e embedded in a type I collagen matrix and cultured in serum-free medi
um. Collagenolytic activity of the cells was determined by measuring t
he amount of hydroxyproline released into the medium from degraded col
lagen. Activities of collagenase in the medium were also measured, usi
ng collagen labeled with fluorescein isothiocyanate as a substrate. Th
e presence of plasminogen was required for collagen degradation by ker
atocytes. In the presence of plasminogen, the amount of collagen degra
dation depended on both the cultivation period and the number of cells
. The addition of interleukin-l (IL-1) stimulated the collagen degrada
tion in a dose-dependent manner. This stimulatory effect of IL-1 was c
ompletely inhibited by the addition of IL-1 receptor antagonist (IL-1r
a). Collagenase activity in the medium was stimulated by the addition
of IL-1, and IL-1ra antagonized this stimulatory effect. These finding
s indicate that our present model may be useful for investigating the
mechanism of collagen degradation by keratocytes. (C) 1998 Japanese Op
hthalmological Society.