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Clinical and Diagnostic Laboratory Immunology, May 1999, p. 437-439, Vol. 6, No. 3
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Inhibition of the Activities of Matrix
Metalloproteinases 2, 8, and 9 by Chlorhexidine
Renée
Gendron,1
Daniel
Grenier,1,*
Timo
Sorsa,2 and
Denis
Mayrand1
Groupe de Recherche en Écologie
Buccale, Faculté de Médecine Dentaire, Université
Laval, Québec, Canada,1 and
Department of Periodontology, University of Helsinki, Helsinki,
Finland2
Received 8 September 1998/Returned for modification 4 December
1998/Accepted 19 January 1999
 |
ABSTRACT |
Matrix metalloproteinases (MMPs) are a host cell-derived
proteolytic enzyme family which plays a major role in
tissue-destructive inflammatory diseases such as periodontitis. The aim
of the present study was to evaluate the inhibitory
effect of chlorhexidine (CHX) on MMP-2 (gelatinase A), MMP-9
(gelatinase B), and MMP-8 (collagenase 2) activity. Heat-denatured
type I collagen (gelatin) was incubated with pure human MMP-2 or -9 activated with p-aminophenylmercuric acetate (APMA), and
the proteolytic degradation of gelatin was monitored by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue
staining. The effect of CHX on MMP-8 activity was also studied with a
cellular model addressing the ability of phorbol
myristate acetate (PMA)-triggered human peripheral blood neutrophils (polymorphonuclear leukocytes [PMNs]) to degrade native type I collagen. CHX inhibited the activities of both
gelatinases (A and B), but MMP-2 appeared to be more sensitive
than MMP-9. Adding calcium chloride to the assay mixtures almost
completely prevented the inhibition of MMP-9 activity by CHX, while the
inhibition of MMP-2 activity could be reversed only when CHX was used
at a low concentration. This observation suggests that CHX may act via
a cation-chelating mechanism. CHX dose-dependently
inhibited collagenolytic activity of MMP-8 released by PMA-triggered
PMNs. MMP-8 without APMA activation was inhibited clearly more
efficiently than APMA-activated MMP-8. Our study
suggests that the direct inhibition of the MMPs' activities by
CHX may represent a new valuable effect of this
antimicrobial agent and explains, at least in part, the
beneficial effects of CHX in the treatment of periodontitis.
| |
TEXT |
Periodontal diseases are
inflammatory conditions which gradually lead to impairment and
destruction of the tooth's supportive tissues. The extracellular
matrix components, including collagens, fibronectin, and proteoglycans,
are the major tissue proteins responsible for the structural integrity
of the tooth's anchoring apparatus. Destruction of the supportive
apparatus is characterized by a degradation of the extracellular matrix
components, leading to irreversible loss of periodontal soft connective
tissues and alveolar bone. Bacterial pathogens and their products are
the primary etiologic agents that directly initiate periodontal disease (11). However, it is the host inflammatory and immune
response, triggered by these pathogens and their virulence
factors, that is mainly responsible for the tissue destruction observed
during periodontitis (18). There is an increasing body
of evidence that suggests that a family of host-derived proteolytic
enzymes called matrix metalloproteinases (MMPs) plays a major role in this pathological process (3, 19, 23). Hence, MMP inhibition would be a valuable adjunct to any periodontal treatment. A wide variety of products are presently being investigated as MMP
inhibitors (1). For instance, tetracyclines and their
chemically modified derivatives have been shown to inhibit the activity
of several MMPs (6-8). Interestingly, it has been
demonstrated that low-dose doxycycline regimens inhibit pathologically
elevated collagenase activity in subgingival sites and prevent the
progression of periodontitis (5). Also, certain
bisphosphonates can inhibit the activity of several human MMPs
(28, 29).
Chlorhexidine (CHX) is a widely used antimicrobial agent that possesses
a broad spectrum of activity against oral bacteria (27).
This cationic bis-biguanide has a unique effect of dental plaque
inhibition, mainly due to its substantivity and its antimicrobial property. It has been demonstrated that CHX mouth rinses are an efficient adjunct to periodontal therapy by controlling supragingival plaque and gingival inflammation (20). Other studies have
confirmed that subgingival irrigation with CHX reduces periodontal
inflammation, sulcate bleeding, periodontal pocket depth, and
subgingival plaque (16, 34). Consequently, CHX mouth
rinses are widely used in the prophylaxis and treatment of
periodontal diseases.
Although numerous beneficial in vivo and in vitro effects of CHX have
been reported, the mechanism of action of this compound continues to be
the subject of numerous investigations as it has not been completely
elucidated. Among its interesting properties, CHX has shown an
antioxidative capacity (4) as well as an ability to
significantly reduce the adherence of Porphyromonas
gingivalis (9) and the proteolytic activity of a number
of periodontal pathogens (2, 10). However, no data
concerning the effect of CHX on host proteases present in affected
periodontal sites are currently available. The aim of the present
study was thus to evaluate the inhibitory effect of therapeutically
attainable concentrations of CHX on the activities of MMP-2, -8, and
-9, as a new beneficial effect in the treatment of periodontitis. These three MMPs were selected because they have been suggested to participate in tissue destruction during periodontitis (14, 15,
21, 24, 25).
Materials and methods. (i) Inhibition assay for MMP-2 (gelatinase
A) and MMP-9 (gelatinase B) activity.
Two genetically distinct
pure human gelatinases were used: MMP-2 (72-kDa gelatinase A isolated
from human fibrosarcoma cells and purchased from Boehringer Mannheim,
Montréal, Québec, Canada) and MMP-9 (human recombinant
92-kDa gelatinase B isolated from mammalian cells and purchased from
Oncogene Research Products, Cambridge, Mass.). MMP-2 was used at a
concentration of 15 U/ml in 50 mM NaCl-20 mM Tris-HCl, pH 8.3, whereas
the MMP-9 was used at a concentration of 20 µg/ml in 5 mM Tris-HCl,
pH 7.5, containing 0.1 mM CaCl2, 0.005% Brij 35, and 10%
glycerol. Both gelatinases were activated by incubation at 37°C for
30 min in 0.5 mM p-aminophenylmercuric acetate (APMA) in a
buffer containing 50 mM Tris-HCl, pH 7.5, and 50 mM NaCl (and 0.005%
Triton X-100 for MMP-2). The effects of CHX on MMP-2 and -9 activity
were tested as follows. Activated MMP-2 (3 µl) or -9 (5 µl) was
incubated with 10 µl of the CHX solution and 5 µl of 50 mM
Tris-HCl, pH 7.5, for 30 min at room temperature. Final concentrations
of CHX diacetate salt (Sigma Chemical Co., St. Louis, Mo.) were 0.03, 0.015, 0.008, 0.004, 0.002, and 0.0001%, prepared in distilled water.
Thereafter, 10 µl of gelatin at 0.5 mg/ml was added, and the assay
mixtures were further incubated at 37°C for 18 h. Gelatin was
prepared by treating type I collagen at 60°C for 30 min. The final pH
of the assay mixtures was 7.5. EDTA at 10 mM was used as a control for
inhibition of MMP-2 and -9 activity. Following incubation, the assay
mixtures were heated at 60°C for 30 min in the presence of
electrophoresis sample buffer and subjected to sodium dodecyl
sulfate-7.5% polyacrylamide gel electrophoresis (SDS-PAGE). Proteins
were stained with Coomassie brilliant blue R-250. The inhibitory effect
of CHX was also tested in the presence of 0.75 mM CaCl2 in
the assay mixtures.
(ii) Inhibition assay for MMP-8 (collagenase 2) activity released
by triggered human neutrophils.
Human neutrophils were used as a
source of MMP-8. Human neutrophils (polymorphonuclear leukocytes
[PMNs]) from peripheral blood were prepared according to the
method of Konttinen et al. (17), adjusted to
1.5 × 107/ml in Hanks balanced salt solution, and
treated for 30 min at 37°C with 50 ng of phorbol
myristate acetate per ml. The cells were pelleted, and
the supernatant containing MMP-8 activity was treated or not with
either 0.005, 0.01, or 0.02% CHX and incubated with 1.5 µM native
type I collagen at 3 mg/ml in 50 mM Tris-HCl (pH 7.4)-0.2 M
NaCl-0.5 mM CaCl2 for 12 h at 22°C. Collagen
cleavage products were analyzed by SDS-10% PAGE and stained by
Coomassie brilliant blue R-250. These experiments were performed
in the absence and presence of 1 mM APMA to activate latent MMP-8 activity.
Results and discussion.
Under the assay conditions used,
pure human MMP-2 and -9 completely degraded the gelatin,
whereas no degradation occurred when the chelating agent EDTA was
included during the incubation. At a concentration of 0.03%, CHX
produced a complete inhibition of MMP-2 and -9 gelatinase activities
(Fig. 1, lanes 4). The inhibitory effect
of CHX was concentration dependent. The minimal concentration of CHX
that led to a complete inhibition of MMP-9 activity was 0.002%,
whereas MMP-2 activity was much more sensitive, being inhibited at a
CHX concentration as low as 0.0001% (data not shown). Calcium chloride
was added to the assay mixtures to verify whether a calcium-chelating
mechanism might be involved in the inhibitory properties of CHX. Adding
calcium chloride to the assay mixtures that contained 0.03% CHX almost
completely prevented the inhibition of MMP-9 activity (Fig. 1B, lane 5)
but had no effect on the inhibition of MMP-2 activity (Fig. 1A,
lane 6). However, decreasing the concentration of CHX to 0.004%
allowed partial reversibility of the inhibition of MMP-2 activity by
addition of calcium chloride (Fig. 1A, lane 7).
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FIG. 1.
Effect of CHX on MMP-2 and -9 activity. MMPs activated
with APMA, CHX, and gelatin were incubated for 18 h at 37°C.
Gelatin degradation was monitored by SDS-7.5% PAGE and Coomassie blue
staining. (A) Lanes: 1, gelatin alone; 2, MMP-2 and gelatin; 3, MMP-2, gelatin, and 10 mM EDTA; 4, MMP-2, gelatin, and 0.03% CHX; 5, MMP-2, gelatin, and 0.004% CHX; 6, MMP-2, gelatin, 0.03% CHX, and
0.75 mM calcium chloride; 7, MMP-2, gelatin, 0.004% CHX, and 0.75 mM
calcium chloride. (B) Lanes: 1, gelatin alone; 2, MMP-9 and gelatin; 3, MMP-9, gelatin, and 10 mM EDTA; 4, MMP-9, gelatin, and 0.03% CHX; 5, MMP-9, gelatin, 0.03% CHX, and 0.75 mM calcium chloride. Molecular
mass markers were, from top to bottom, myosin (200 kDa), phosphorylase
b (97.4 kDa), and bovine serum albumin (68 kDa).
|
|
The activity of MMP-8 released by phorbol myristate
acetate-triggered human neutrophils and without the APMA
activation treatment
was completely inhibited by CHX at
concentrations of 0.02 and
0.01% (Fig.
2). The MMP-8 collagenase activity
present in the
PMN supernatant and activated by APMA was only partially
inhibited
by the same concentrations of CHX.
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FIG. 2.
Effect of CHX on autoactivation of MMP-8 activity. Human
neutrophil supernatant, CHX, and collagen were incubated for 12 h
at 22°C. The assays were carried out in the absence or presence of 1 mM APMA to activate the MMP-8 (collagenase 2) activity. Collagen
degradation was monitored by SDS-10% PAGE and Coomassie blue
staining.  , intact -chains of type I collagen; A,
characteristic three to four cleavage products resulting from
collagenase action. Lanes: 1, type I control collagen; 2, type I
collagen and neutrophil supernatant; 3, type I collagen, neutrophil
supernatant, and 0.005% CHX; 4, type I collagen, neutrophil
supernatant, and 0.01% CHX; 5, type I collagen, neutrophil
supernatant, and 0.02% CHX; 6 to 9, same as lanes 2 to 5, respectively, except that 1 mM APMA was present.
|
|
Considerable evidence supports the importance of MMPs as key host
cell-derived mediators of the pathological tissue destruction
observed
during periodontitis (
3,
19,
23). More particularly,
MMP-2,
-8 and -9 in active forms have been found at pathologically
elevated
levels in saliva, gingival crevicular fluid, and gingival
tissues of
periodontitis patients (
7,
14,
15,
21,
25).
The potential
cellular sources of MMP-2 and -9 include resident
gingival and
periodontal ligament fibroblasts, as well as endothelial
and epithelial
cells. In addition, MMP-9 in periodontal inflammation
can originate
from triggered neutrophils, which are also major
sources of MMP-8.
Furthermore, recent studies have shown that
certain non-PMN lineage
cells, such as gingival and periodontal
ligament fibroblasts and
endothelial cells, can express MMP-8
(
12,
25). Overall,
multiple resident and inflammatory host
cellular sources may be the
origin of the MMPs studied for the
inhibitory effect of CHX. Based on
these observations, MMP inhibitors
are being investigated as a new
class of potential therapeutic
agents in the treatment of
periodontitis. As a matter of fact,
inhibition of MMP activity by
tetracyclines and their chemically
modified derivatives has shown
promising results (
5,
7,
25).
The present study has shown that CHX directly inhibits
MMP-2, -8, and -9 activity. MMP-2 appeared to be more sensitive
than
MMP-8 and -9. A chelating mechanism might be involved in the
inhibition
of MMP-2 and -9, since adding calcium chloride to the assay
mixture
prevented the inhibition of these enzymes by CHX.
However, the
effect of an excess of calcium chloride was not identical
for
MMP-2 and -9; the inhibition of MMP-2 activity was reversed
only
when CHX was used at a low concentration (0.004%). It is likely
that at high concentrations of CHX, MMP-2 is inactivated by protein
denaturation (
13) rather than by the chelation of cations.
It
has been previously proposed that tetracyclines inhibit MMP activity
by their cation-binding properties (
6), since MMPs require
metal ions, namely, calcium and zinc, for their catalytic
activity.
Dichloromethylene biphosphonate (clodronate),
which has been shown
to inhibit MMP-1 and -8, is also thought to
act as a cation chelator
(
28,
29).
Additionally, it was found that CHX (0.01 and 0.02%) completely
inhibited autoactivated MMP-8, in comparison to a poor inhibition
of
APMA-activated MMP-8, released by triggered human PMNs. APMA,
an
organomercurial MMP activator capable of attacking protein
sulfhydryl
groups or inducing cysteine switch reactions involved
in both the
activation and inhibition of MMP-8 (
30-32), was found
to
prevent the CHX inhibition of MMP-8. This suggests that CHX
may
interact with the essential sulfhydryl groups and/or cysteine
present
in the active site of MMPs. Regarding the molecular pathogenesis
of
periodontitis, it is worthy of note that CHX efficiently inhibited
impure endogenously activated MMP-8 in the presence of other
biologically
active molecules and inflammatory mediators released by
triggered
PMNs (
33), since it has been frequently shown that
in active
periodontitis lesions, i.e., gingival tissues and adjacent
crevicular
fluid (
19,
22), MMP-8 is converted from a latent
form to an
active form (
25).
CHX is a valuable agent for the treatment of periodontal
diseases and can be used in mouth rinses or other local delivery
methods (
26). The inhibition of MMPs by CHX demonstrates new
beneficial antiproteolytic properties which, in addition to the
known
antimicrobial properties of this substance, are useful in
the treatment
of periodontitis. Clinical trials are now in progress
to further
evaluate these new anti-MMP properties in
vivo.
| |
ACKNOWLEDGMENTS |
This work was supported by the Fonds Emile-Beaulieu.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Groupe de
Recherche en Écologie Buccale, Faculté de Médecine
Dentaire, Université Laval, Cité Universitaire,
Québec, Canada G1K 7P4. Phone: (418) 656-7341. Fax: (418)
656-2861. E-mail: Daniel.Grenier{at}greb.ulaval.ca.
| |
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Clinical and Diagnostic Laboratory Immunology, May 1999, p. 437-439, Vol. 6, No. 3
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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