A novel in-situ-zymography technique localizes gelatinolytic activity in human skin to mast cells.

Matrix-metallo-proteinases play a key role in cutaneous tissue remodeling and wound healing, and have been implicated as the rate-limiting factor in cutaneous tumor invasion and metastasis. We here describe a novel in-situ-zymographic method, which allows to directly localize sites of gelatinolytic activity in human skin. Gelatinolysis was detected through protein-hydrolysis in a 200 microm thick polyacrylamide gel underlying tissue sections. The lysis was substrate-dependent, demonstrated time- and temperature-dependent kinetics, and was inhibited by both EDTA and 1,10-phenanthroline. Normal and diseased skin sections demonstrated multiple focal points of gelatinolysis which co-localized with individual cells. Histochemically, these were shown to represent most likely mast cells (via AS-d-chloroacetate esterase staining and metachromasia). However, immunohistochemical staining for gelatinases A and B showed no immunoreactivity patterns that corresponded to the identified foci of gelatinolysis. The reported in-situ-zymographic technique offers a decisive advantage over immunohistochemistry, since it detects only the activated and catabolically relevant proteases, and provides further evidence for a role of mast cells in extracellular matrix remodeling.