INVESTIGATIONS INTO THE CELLULAR CONTRIBUTION TO HOST TISSUE PROTEASES AND INHIBITORS IN GINGIVAL CREVICULAR FLUID (CORRECTED VERSION OF XE471)

Gingival crevicular fluid (GCF) was collected from chronic periodontit is patients using plastic micropipettes and coverslip smears stained w ith antibodies for leukocyte markers and Toluidine Blue for mast cells . The smears consisted of 70-80% granulocytes, 10-20% monocytes/macrop hages, 5% mast cells and 5% T lymphocytes; no B lymphocytes were found . Proteases and inhibitors in GCF cells were investigated by enzyme cy tochemistry using 2-methoxy-4-naphthylamine-linked peptide substrates and simultaneous coupling to Fast Blue B and immunocytochemistry using biotinylated secondary antibodies and an alkaline phosphatase/new fuc hsin detecting system. Elastase was detected in granulocytes, cathepsi n B in macrophages, dipeptidyl peptidases II and IV in a small proport ion of macrophages, dipeptidyl peptidase IV in a few T lymphocytes, tr yptase in mast cells and alpha-1-proteinase inhibitor and alpha-2-macr oglobulin in some macrophages. GCF was also collected on filter paper strips and eluted into buffer for biochemical enzyme assays. Lysis of cells by addition of detergent to the elution buffer increased activit ies to 140-240% of control values. Removal of cells by centrifugation reduced measured activities to 1-30% of original figures; this effect was less if samples were pre-treated with detergent. Proteases from in flammatory cells therefore appear to make up most of the measured enzy me activity in GCF, and this association may explain recent correlatio ns with periodontal disease progression.