Doxycycline inhibits neutrophil (PMN)-type matrix metalloproteinases in human adult periodontitis gingiva. J Clin Periodontol 1995; 22: 100^109. © Mutiksgaard, i995.Abstract We previousiy reported tliat low-dose doxycycline (DOXY) therapy reduces host-derived coliagenase activity in gingival tissue of aduit periodontitis (AP) patients. However, it was not ciear wiiether this in vivo effect was direct or indirect. In the present study, iniiamed human gingival tissue, obtained from AP patients during periodontal surgery, was extracted and the extracts partia!!y purified by (NH4)2SO4 precipitation. The extracts were then ana!yzed for coUagenase activity using SDS-PAGE/iluofography/iaser densitometry, and for geiatinase activity using type I geiatin zymography as weii as a new quantitative assay using biotinyiated type I geiatin as substrate. DOXY was added to the incubation mixture at a fina! concentration of 0-!000 ^M. The concentration of DOXY required to inhibit 50% of the gingiva! tissue coiiagenase (IC50) was found to be i6-18 fiM in the presence or absence of i.2 mM APMA (an optimai organomercurial activator of latent procoiiagenases); this IC50 for DOXY was simiiar to that exhibited for coiiagenase or matrix metaiioproteinase (MMP)-8 from polymorphonuclear leukocytes (PMNs) and from gingival crevicuiar fluid (GCF) of AP patients. Of interest. Porphyromonas gingivalis collagenase was also inhibited by similar DOXY levels (IC5o=15 /^M), however the collagenase activity observed in the gingival tissue extracts was found to be of mammalian not bacterial origin based on the production of the specific a'^ (3/4) and a^ (i/4) coliagen degradation fragments. In contrast, the inhibition of coiiagenase purified from cuiture media of human gingival fibroblasts (MMP-1) required much greater DOXY levels (IC5o=280 //M). The predominant molecular forms of gelatinolytic activity present in the AP patients gingivai tissue extracts were found to closeiy correspond to the 92 kD PMN-type geiatinase (MMP-9) although small quantities of 72 kD fibrobiast-type geiatinase (MMP-2), and some other iow moiecuiar weight geiatinases, were also detected. The IC50 of DOXY versus gingivai tissue gelatinoiytic activity was estimated at 30-50 //M measured using either type I geiatin zymography or the biotinylated type I gelatin assay. We conclude that MMPs in inflamed gingival tissue of AP patients, iike those in GCE, originate primarily from infiltrating PMNs rather than resident gingival cells (fibroblasts and epitheha! ce!!s) or monocyte/macrophages, and that their patho!ogica!!y-elevated tissue-degrading activities can be directly inhibited by pharmaco!ogic !eve!s of doxycyc!ine.

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