DOXYCYCLINE INHIBITS NEUTROPHIL (PMN)-TYPE MATRIX METALLOPROTEINASES IN HUMAN ADULT PERIODONTITIS GINGIVA

We previously reported that low-dose doxycycline (DOXY) therapy reduce s host-derived collagenase activity in gingival tissue of adult period ontitis (AP) patients. However, it was not clear whether this in vivo effect was direct or indirect. In the present study, inflamed human gi ngival tissue, obtained from AP patients during periodontal surgery, w as extracted and the extracts partially purified by (NH4)(2)SO4 precip itation. The extracts were then analyzed for collagenase activity usin g SDS-PAGE/fluorography/laser densitometry, and for gelatinase activit y using type I gelatin zymography as well as a new quantitative assay using biotinylated type I gelatin as substrate. DOXY was added to the incubation mixture at a final concentration of 0-1000 mu M. The concen tration of DOXY required to inhibit 50% of the gingival tissue collage nase (IC50) was found to be 16-18 mu M in the presence or absence of 1 ,2 mM APMA (an optimal organomercurial activator of latent procollagen ases); this IC50 for DOXY was similar to that exhibited for collagenas e or matrix metalloproteinase (MMP)-8 from polymerphonuclear leukocyte s (PMNs) and from gingival crevicular fluid (GCF) of AP patients. Of i nterest, Porphyromonas gingivalis collagenase was also inhibited by si milar DOXY levels (IC50=15 mu M), however the collagenase activity obs erved in the gingival tissue extracts was found to be of mammalian not bacterial origin based on the production of the specific alpha(A) (3/ 4) and alpha(B) (1/4) collagen degradation fragments. In contrast, the inhibition of collagenase purified from culture media of human gingiv al fibroblasts (MMP-1) required much greater DOXY levels (IC50 = 280 m u M). The predominant molecular forms of gelatinolytic activity presen t in the AP patients gingival tissue extracts were found to closely co rrespond to the 92 kD PMN-type gelatinase (MMP-9) although small quant ities of 72 kD fibroblast-type gelatinase (MMP-2), and some other low molecular weight gelatinases, were also detected. The IC50 of DOXY ver sus gingival tissue gelatinolytic activity was estimated at 30-50 mu M measured using either type I gelatin zymography or the biotinylated t ype I gelatin assay. We conclude that MMPs in inflamed gingival tissue of AP patients, like those in GCF, originate primarily from infiltrat ing PMNs rather than resident gingival cells (fibroblasts and epitheli al cells) or monocyte/macrophages, and that their pathologically-eleva ted tissue-degrading activities can be directly inhibited by pharmacol ogic levels of doxycycline.