IMMUNOLOCALIZATION OF COLLAGENASE IN RABBIT PERIOSTEAL TISSUE EXPLANTS AND EXTRACTION OF THE ENZYME - THE EFFECT OF THE CYTOKINES IL-1-ALPHA AND EGF

The effect of interleukin-1 alpha (IL-1 alpha) and murine epidermal gr owth factor (EGF) on incorporation of endogenously produced collagenas e in the extracellular matrix of soft connective tissue was studied in an in vitro model system using periosteal explants obtained from rabb it calvariae. Immunohistochemical analysis indicated the highest level of collagenase in explants cultured for 72 hours with IL-1 alpha in c ombination with EGF. Most enzyme appeared to be associated with the ex tracellular matrix, but labeling was also found in numerous fibroblast -like cells. Explants cultured in the presence of IL-1 alpha alone con tained less enzyme and in periostea treated without cytokines, or with EGF alone, only a faint label, if any, was seen. Freshly isolated, no n-cultured periostea contained no detectable enzyme. Extraction of col lagenase from periostea revealed that: (1) non-cultured periosteum did not contain detectable levels of enzyme. (2) The amount of total acti vatable enzyme synergistically increased (10-fold) under the influence of IL-1 alpha and EGF, whereas IL-1 alpha alone showed a 4-fold enhan cement compared to control or EGF-incubated explants. (3) The latent f raction of the enzyme was synergistically increased (up to 100-fold or more) in periostea cultured in the presence of IL-1 alpha+EGF (21.17 mU/explant versus 0.05 mU/explant in controls). (4) Active collagenase , on the other hand, appeared to be present in a relatively high conce ntration in explants cultured without cytokines (2.45 mU/explant versu s 0.36 mU/explant in IL-1 alpha+EGF-treated explants). (5) No enzyme c ould be extracted from devitalized explants that were incubated in med ia containing high levels of latent collagenase. It was concluded that collagenase had been incorporated somehow in the extracellular matrix during culturing. In an attempt to analyze breakdown of collagen the amount of hydroxyproline in culture media was assessed. The level of t his imino acid released by periostea treated without cytokines or with EGF alone was significantly higher than by those incubated with IL-1 alpha and EGF. In conclusion, incorporation of collagenase in perioste al connective tissue seems to occur during culturing; the level of the incorporated latent enzyme being strongly enhanced by a combination o f the cytokines IL-1 alpha and EGF. However, as the amount of collagen breakdown had decreased, these findings indicate that PL-1 alpha in c ombination with EGF is able to induce deposition of a large reservoir of latent collagenase in the extracellular matrix, but does not stimul ate adequate activation mechanisms to result in enhanced degradation.