COMPARATIVE HISTOCHEMICAL, BIOCHEMICAL AND IMMUNOCYTOCHEMICAL STUDIESOF CATHEPSIN-B IN HUMAN GINGIVA

Cathepsin B activity was demonstrated histochemically in unfixed cryos tat sections of inflamed human gingiva using the 2-methoxy-4-naphthyla mide (MNA) substrates Z-Val-Lys-Lys-Arg-MNA and Z-Ala-Arg-Arg-MNA with a post-azo-coupling technique. Enzyme localisation was confirmed by i mmunocytochemistry with polyclonal sheep anti-human cathepsin B. In bo th cases, staining was found in connective tissue fibroblasts and also in cells varying in shape from rounded to more irregular forms. The l atter were present both in areas of cellular infiltration and in the o ral and pocket epithelium. Examination of adjacent sections with monoc lonal antibodies directed against leukocyte differentiation antigens s howed that the rounded to irregular cells were CD68 positive macrophag es and monocytes. The histochemical staining had the form of fine cyto plasmic particles consistent with the known lysosomal occurrence of ca thepsin B. Cells stained by the post-coupling method using the tryptas e substrates Z-Ala-Ala-Lys-MNA and D-Val-Leu-Arg-MNA showed a differen t distribution and morphology, with reaction product confined to mast cell granules. The differences between the cathepsin B and tryptase st aining patterns were confirmed by differential extraction from cryosta t sections with salt-free and high-salt buffers respectively. Biochemi cal characterisation of activities in the extracts with the 7-amino-4- trifluoromethyl coumarin (AFC) substrates Z-Val-Lys-Lys-Arg-AFC and Z- Ala-Ala-Lys-AFC and protease inhibitors confirmed the identity of the two enzymes. Selective inhibitors could also be used in histochemical incubations to distinguish between cathepsin B and tryptase staining.