A NOVEL IN-SITU-ZYMOGRAPHY TECHNIQUE LOCALIZES GELATINOLYTIC ACTIVITYIN HUMAN SKIN TO MAST-CELLS

Matrix-metallo-proteinases play a key role in cutaneous tissue remodel ing and wound healing, and have been implicated as the rate-limiting f actor in cutaneous tumor invasion and metastasis. We here describe a n ovel in-situ-zymographic method, which allows to directly localize sit es of gelatinolytic activity in human skin. Gelatinolysis was detected through protein-hydrolysis in a 200 mu m thick polyacrylamide gel und erlying tissue sections. The lysis was substrate-dependent, demonstrat ed time- and temperature-dependent kinetics, and was inhibited by both EDTA and I,10-phenanthroline. Normal and diseased skin sections demon strated 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 metac hromasia). However, immunohistochemical staining for gelatinases A and B showed no immunoreactivity patterns that corresponded to the identi fied foci of gelatinolysis. The reported in-situ-zymographic technique offers a decisive advantage over immunohistochemistry, since it detec ts only the activated and catabolically relevant proteases, and provid es further evidence for a role of mast cells in extracellular matrix r emodeling.