Cn. Kennett et al., INVESTIGATIONS INTO THE CELLULAR CONTRIBUTION TO HOST TISSUE PROTEASES AND INHIBITORS IN GINGIVAL CREVICULAR FLUID, Journal of clinical periodontology, 24(6), 1997, pp. 404-411
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 TV 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.