Inflammatory injury to the gingival stroma in periodontal disease is mediated in part by serine proteases and matrix metalloproteases (MMPs) released by neutrophils and macrophages recruited to sites of bacterial overgrowth. We propose to study interactions of two of the most potent of these proteases, neutrophil elastase (NE), and neutrophil collagenase (MMP- 8), in mediating injury to interstitial connective tissue. We will investigate how tetracycline (TCs), which have been used therapeutically in controlling periodontal disease, might affect these interacting proteases. Instead of animal models of periodontitis, we will employ a complete biosynthetically labeled interstitial extracellular matrix from cultured R22 rat smooth muscle cells (R22 ECM) for in vitro studies on interactions of purified proteases with natural antiproteases or synthetic inhibitors, for measurements of leukocyte-mediated connective tissue degradation, and for observations of leukocyte invasive migration. In collaboration with Dr. L.M. Golub at Stony Brook, we will evaluate the capacity of several chemically modified TCs with no antibiotic activity to inhibit degradation of R22 ECM by purified proteases or by activated leukocytes. We have reported that reaction of NE with its major endogenous inhibitor alpha1- antitrypsin (alpha1-PI) to ECM with retention of antiprotease activity. We propose to extend these studies to PAI-1, another SERine Protease inhibitor (serpin) which inhibits macrophage urokinase and can also bind to interstitial ECM. A significant portion of the inactivation of ECM-bound serpins reflects proteolytic cleavage by MMPs of leukocyte and interstitial origin. We will pursue initial results which show that TCs can inhibit proteolytic inactivation of both fluid phase and ECM-bound alpha1-PI, as well as other serpins, by stimulated leukocytes and their MMPs, and by ECM- associated MMP activity. Such inhibition may help to preserve the antiprotease activity of the serpins, and may be a major mechanism by which TCs reduce inflammatory tissue damage. The role of proteases in migration of leukocytes to inflammatory foci is still controversial. We will use a model we have developed for invasive migration of leukocytes through the R22 ECM on porous plastic membrane-bottomed cell culture inserts to study the effects of alpha1-PI, PAI-1, and the TCs, separately and in combination, on invasion of neutrophils and macrophages through an interstitium like that of the gingival stroma, which we will correlate with inhibition of ECM degradation.