Figure 5.

Effect of mMCP-4 on progelatinase B present in hyperplastic tissue and localization of mMCP-4 and gelatinase B in vivo. (A) Gelatinolytic activity in tissue lysates (2 μl) from normal (N), hyperplastic (H), dysplastic (D) and carcinoma (T) biopsies. Incubation of gelatin zymograms with 1,10 phenanthroline (an inhibitor of MMPs), but not PMSF (an inhibitor of serine proteases) following electrophoresis abolished the 68, 72, 80, and 90-kD bands completely (data not shown). (B) In vitro reconstitution of gelatinase B activity. Two microliters of hyperplastic (H) lysate alone or 2 μl of hyperplastic lysate incubated for 30 min at 37°C with 40 ng of purified mMCP-4. Molecular masses are shown in kD. (C–D) Immunolocalization of mMCP-4 (red staining) and laminin (brown staining) at basement membranes (bm, arrows) adjacent to epithelium (e) and capillaries (c) in dysplastic skin. (E) Immunolocalization of gelatinase B (blue staining) in dysplastic skin localizes to basement membranes (bm) adjacent to epithelium (e, closed arrows) and around capillaries (c, open arrows) in dermis. (F) Control for nonspecific binding using a control rabbit IgG; background staining was negligible. Bar, 44.6 μm (C–F).