Abstract
The purpose of this experiment was to study activities of dipeptidyl peptidase IV (DP-IV) and collagenase-like peptidase (CLP) released from macrophages by stimulation of Bacteroides gingivalis in vitro.
Considerable evidence has been accumulated that cellmediated immune mechanisms may be involved in the initiation and peogression of periodontal diseases. Macrophages are widely distributed in connective tissues, organs and periodontal tissues. Macrophages may play a central role in inducement and appearance of immunity against bacterial plaque.
It is a remarkable fact that macrophages in inflammatory lesions may have the function not only of self-defense by phagocytosis but also of destruction of connective tissue by released hydrolytic enzymes. On the other hand, B. gingivalis has been recently recognized as a pathogenic microorganism in periodontal diseases. It is reported that B. gingivalis is predominantly isolated from periodontal pocket in some advancing lesions of adult periodontitis. It has been also found that a strong correlation exists between the levels of serum antibody against B. gingivalis strain and periodontal diseases.
As an in vitro experiment, peritoneal macrophages were obtained from 250-300g male rats (Std: Donryu) by irrigating the peritoneum with RPMI1640 media. Macrophages in culture wells were purified by removing non-adherent cells by washing with the media after incubation. B. gingivalis cells, opsonized B. gingivalis cells, latex beads (φ0.81μm), and media were added in macrophage (0.5×106cells) culture wells. Incubated culture media-an enzyme solution-were collected at variable stimulated volumes and time intervals.
The activities of DP-IV and CLP were measured using Gly-Pro-MCA, Suc-Gly-Pro-Leu-Gly-Pro-MCA as substrates by measuring liberated AMC from substrates. The digested products of the action of released DP-IV from macrophages on partially digested peptide from collagen type I as substrate were analyzed using high-pressure liquid chromatography.
Activities of DP-IV and CLP increased against stimulated volumes and maximum activities at stimulated concentration were 0.5×10 cells. Those of latex beads groups were approximately the same as the control media. In the opsonized cell groups, maximum activity of DP-IV was observed after 12hrs. and that of CLP 4 hrs. after stimulation. Both of the enzyme activities of opsonized cell groups were 2.5-times against non-treated cell groups. The enzyme properties were as follows: The optium pH for DP-IV activity was 8.6 using tris-maleate buffer and that for CLP activity was 8.2 using tris-HCl buffer. The Km value of DP-IV was 0.067mM using Gly-Pro-MCA as a substrate and that of CLP was 0.011mM using Suc-Gly-Pro-Leu-Gly-Pro-MCA as substrate. It seemed that neither inhibitor nor acti vator were present in this enzyme solution with DPIV and CLP. This enzyme solution hydrolyzed partially digested peptide from collagen type I and liberated Gly-Pro peptide.
The enzymes of DP-IV and CLP were released from macropahges by stimulation of B. gingivalis and the release was encouraged by opsonization.
