COLLAGENOLYTIC ACTIVITY OF KERATOCYTES CULTURED IN A COLLAGEN MATRIX

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.