INFECTION AND IMMUNITY, Dec. 1991, p. 4687-4692 Vol. 59, No.

12
0019-9567/91/124687-06$02.00/0
Copyright © 1991, American Society for Microbiology

Evidence for Polymorphonuclear Leukocyte Collagenase and

92-Kilodalton Gelatinase in Gingival Crevicular Fluid
CHRISTOPHER M. OVERALL,'* JARO SODEK,2 CHRISTOPHER A. G. McCULLOCH AND PETER BIREK3 ,

Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia,' and Medical Research Council
Group in Periodontal Physiology2 and Faculty of Dentistry,3 University of Toronto, Toronto, Ontario, Canada
Received 17 April 1991/Accepted 25 September 1991
Analysis of inflammatory exudate collected from sites of experimental periodontitis in cynomolgus monkeys
has revealed the presence of coliagenase and a 92-kDa gelatinase that comigrated after electrophoresis with the
92-kDa gelatinase released from polymorphonuclear leukocytes. Since neutralizing antibodies to fibroblast
collagenase had no effect on the collagenase activity and bacterial collagenases could not be detected, poly-
morphonuclear leukocytes appear to be the major source of collagenolytic proteinases in inflammatory fluid
from gingiva.

Degradation of the extracellular matrix is an important
characteristic of acute and chronic inflammatory lesions, but
the mechanisms of tissue degradation have not been clearly
established (41, 47). The characterization of the proteinases
present in inflammatory exudates can identify the cellular
source of the enzymes and hence indicate the relative
importance of those cells in the degradative processes oc-
curring in the disease. Inflammatory diseases of connective
tissues, such as periodontitis, appear to involve connective
tissue-derived metalloproteinases, such as the interstitial
collagenase of the matrix metalloproteinase (MMP) family
(10, 22, 34, 45), and a distinct collagenase, released only
from polymorphonuclear leukocytes, termed leukocyte col-
lagenase. In periodontitis, collagenases synthesized by
pathogenic microorganisms may also contribute to collagen
loss (5).
Interstitial collagenase (MMP-1) is synthesized by several
cell types present in the periodontium, including gingival (16,
29, 39) and periodontal (29, 32, 40) ligament fibroblasts,
osteoblasts (30, 32), keratinocytes (23), and macrophages
(6). Leukocyte collagenase (MMP-8) is released from storage
granules in polymorphonuclear leukocytes (13, 27), and
although the enzyme cleaves the same scissile bonds in
collagens as interstitial collagenase does, it differs from
interstitial collagenase in its Mr (27), substrate kinetics (14),
and antigenic properties (12). The collagenolytic cascade is
also thought to involve the activity of gelatinases (41) that
can degrade denatured collagens and native type IV colla-
gen. A 72-kDa gelatinase (MMP-2) is synthesized by various
mesenchymal cells (7), including human gingival fibroblasts
(35, 37) and osteoblasts (30, 32, 36), whereas a 92-kDa
gelatinase (MMP-9) is synthesized by polymorphonuclear
leukocytes (27), alveolar macrophages (48), keratinocytes
(48), and some transformed cells (48) but not by normal
fibroblasts (7, 48) or osteoblasts (36). Endothelial cells
produce both forms of gelatinase (26). Bacterial compo-
nents, particularly lipopolysaccharides, are thought to in-
crease MMP expression by inducing the release of inflam-
matory mediators, such as interleukin-1 (25, 40) and tumor
necrosis factor (25), from macrophages (15) and lympho-
cytes; these inflammatory mediators in turn induce MMP
expression in fibroblasts, macrophages, and other resident
*
Corresponding author.
4687
tissue cells. Bacterial cell surface lectins may also directly
induce MMP expression and MMP activation in fibroblasts
(33).
To understand the mechanisms of tissue destruction in
periodontal inflammation, the cellular origin of proteolytic
enzymes and the relative importance of host cells and
bacterial cells as sources of these enzymes need to be
established. Although some indirect evidence indicates that
polymorphonuclear leukocytes are the source of collagenase
in gingival inflammatory exudate (9, 28, 42), this has not
been shown unequivocally. We have addressed this problem
by characterizing the gelatinolytic and collagenolytic en-
zymes in the inflammatory exudate, gingival crevicular fluid,
collected from sites of ligature-induced periodontitis in cyn-
omolgus monkeys (Macaca fascicularis)-an established,
well-characterized model of periodontitis that closely repli-
cates human periodontitis (38). In this model, inflammation
can be induced at will, and the ensuing tissue destruction
occurs in a relatively predictable temporal manner.
Periodontitis was induced in clinically healthy gingiva
around the maxillary incisors of two 4-kg cynomolgus mon-
keys (Connaught Laboratories, Toronto, Ontario, Canada)
by placement of subgingival silk ligatures and withdrawal of
daily prophylaxis for 10 weeks. This method encourages
large increases in numbers of tooth-bound colonies of micro-
organisms. Unligated mandibular incisors served as con-
trols. Full descriptions of the clinical measurements and
indices that were recorded weekly to monitor the loss of the
gingival connective tissue attachment around these teeth and
the development of periodontitis are given in detail else-
where (2). In summary, the microbial load and the amount of
inflammation reached maximum values within 1 week, gin-
gival crevicular fluid flow was significantly increased at 1 and
3 weeks, and there was a progressive loss of connective
tissue attachment around the experimental teeth during the
10-week experiment.
Gingival crevicular fluid was collected in microcapillary
tubes (sample volume, 0.66 + 0.21 ,ul [mean + standard
deviation]) (17), diluted 1/10 with assay buffer (50 mM
Tris-HCl, 200 mM NaCl, 5 mM CaCl2, 0.5 jig of Brij 35 per
ml, 0.2 jig of NaN3 per ml [pH 7.2]), and divided into four
equal aliquots, three of which were used for active and total
collagenase and collagenase inhibitor assays as described
previously (2). The remaining aliquot of the crevicular fluid
4688 NOTES
A B
F 1 2 3 4 5 10 1 2 3 4 5 10
#i,94
.67
-A
43
FIG. 1. Enzymogram analysis of gingival crevicular fluid. On the
enzymograms, gelatinolytic proteinases in gingival crevicular fluid
are revealed as clear bands against the blue-stained gelatin substrate
background. In the examples shown, samples of gingival crevicular
fluid were obtained from two sites of experimental periodontitis (A
and B) collected each week for 5 weeks (lanes 1 to 5) and at week 10
(lane 10) as described in the text. Lane F, 72-kDa gelatinase present
in the conditioned culture medium of human gingival fibroblasts was
electrophoresed nonreduced as a standard. (The 72-kDa gelatinase
migrates with an apparent Mr of 72,000 when electrophoresed under
reducing conditions and with an apparent Mr of 66,000 under
nonreducing conditions.) Arrow A indicates serum albumin in the
crevicular fluid. The positions of migration of Mr marker proteins (in
thousands) and the electrophoresis front (fr) are indicated.
samples was further diluted (1/144) and assayed for gelati-
nolytic and caseinolytic proteinases by enzymography. Sam-
ples (15 ,ul) were electrophoresed under nonreducing condi-
tions on sodium dodecyl sulfate-10% polyacrylamide
separating gels (18) containing either gelatin or casein (40
p.g/ml), and enzymography was performed as described
previously (31). The gelatinolytic and caseinolytic protein-
ases that renatured and were functionally active in the
200-min assays were visualized as cleared bands against the
aqua blue-stained substrate gel. For standards, the condi-
tioned medium of confluent quiescent cultures of human
gingival fibroblasts (Gin-i [CRL 1292]; American Type Cul-
ture Collection, Rockville, Md.) was collected as a source of
fibroblast 72-kDa gelatinase (35). Prestained relative molec-
ular mass marker proteins (Bio-Rad) and the relative molec-
ular mass marker proteins used before (35) were electro-
phoresed under reducing conditions as Mr standards.
At each time point, the gelatin enzymograms revealed the
presence of a gelatinolytic doublet that migrated at 92 and 84
kDa (Fig. 1). In addition, a minor gelatinolytic doublet at 66
and 59 kDa, which comigrated with the human gingival
fibroblast gelatinase, was frequently observed together with
gelatinase activity in the 40-kDa region, at 116 kDa, and of a
very high molecular mass. To assay for stromelysin
(MMP-3) and bacterial proteinases, casein substrate gels
were used, but caseinolytic activity was never detected in
any of the samples, even after prolonged assay incubation
times (24 and 48 h; Fig. 2).
To provide sufficient material for characterization of the
gingival crevicular fluid gelatinases, replicate aliquots of the
diluted crevicular fluid samples, collected site specifically,
were pooled. Samples were incubated with either 1.0 mM
p-aminophenylmercuric acetate (APMA) or solvent at 22°C
for 30 min. The organomercurial APMA activates pro-MMPs
(43) and inhibits thiol proteinases, including the Porphory-
monas gingivalis collagenase (5). Electrophoresis sample
buffer was then added, and the samples were electro-
phoresed on gelatin substrate or casein substrate gels before
incubation in assay buffer. Enzyme activity was compared
by using gels on which identical samples had been electro-
phoresed but each had been incubated either in assay buffer
INFECT. IMMUN.
Conrtrol ED
CTA_
M F p F G GPGPG F pF G G0 p{; Dk *T"-... ED '
>, G F G i'.
3 -9
:3
K3I)
PMSF NEM i, F,; C pG
GCpG pG
F pFG F pF .C pG lp F F ;)
V7
e ' ;ases * ":mo,Itspa,
FIG. 2. Characterization of gingival crevicular fluid gelatinases.
Pooled gingival crevicular fluid samples (lanes G) were electro-
phoresed in duplicate on substrate-polyacrylamide gels containing
40 ,ug of gelatin or casein per ml as described in the text. Proteolytic
activity was assayed by enzymography with assay buffer alone
(Control) or assay buffer containing either 10 mM EDTA, 10 mM
DTT, 10 mM DTT plus 10 mM EDTA, 1 mM PMSF, or 5 mM NEM
as indicated. To activate any progelatinases present in the gingival
crevicular fluid, the samples were treated with 1 mM APMA for 30
min before electrophoresis (lanes pG). As standards, reduced pre-
stained Mr marker proteins (lane M), which electrophoresed with
the apparent Mrs indicated (in thousands), and human gingival
fibroblast 72-kDa gelatinase, both before (lanes F) and after (lanes
pF) APMA activation, were included.
alone or in assay buffer containing one of the following
reagents or combinations of reagents: 5 mM N-ethylmaleim-
ide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10
mM EDTA, 10 mM dithiothreitol (DTT), or 10 mM DTT plus
10 mM EDTA. Human gingival fibroblast 72-kDa gelatinase,
before and after APMA activation, was used as a standard.
APMA caused a decrease in the molecular mass of the
gingival crevicular fluid 92-kDa gelatinase to 84 kDa and a
decrease in the molecular mass of the human gingival
fibroblast 72-kDa gelatinase from 66 kDa under nonreducing
conditions (7, 35) to 62 kDa (Fig. 2). It is notable that the
proenzyme form of gelatinase is activated during electropho-
resis by the sodium dodecyl sulfate (4) present in the
polyacrylamide gels and therefore is also visualized as a
clear band on the enzymograms with a molecular mass
corresponding to the zymogen form of the enzyme. A
134-kDa gelatinase present in the human gingival fibroblast
conditioned medium was absent after APMA treatment and
most likely represents a dimer of the 72-kDa gelatinase. We
have previously observed that APMA activation sometimes
slightly reduces the net activity of collagenase obtained from
gingival crevicular fluid (9, 17), possibly through APMA-
induced autolysis, and that APMA also induces the rapid
autodegradation of rat 72-kDa gelatinase (33a). In this study
too, APMA appeared to reduce the total amount of 92-kDa
gelatinase after activation. However, the thiol blocker NEM
slightly increased gelatinolytic activity, as reported previ-
ously for leukocyte collagenase (44). In addition, the activ-
ities of the crevicular fluid 92-kDa gelatinase and the higher-
VOL. 59, 1991
11 Da. oe!;ai. -ase~
.-
..; KDa CF-laP:naS(,
t: .. i
49* t~~~~~-
g4
t,; S Kl ;z- ^ei
p
23 qr
-
'. -.-
a-e
c4 4 and arylsulfatase have been found in gingival crevicular fluid
Ji
FIG. 3. Affinity purification of polymorphonuclear leukocyte ge-
latinases and comparison with gingival crevicular fluid gelatinases.
The lysate from monkey polymorphonuclear leukocytes (MPMN)
was loaded onto a gelatin-Sepharose column, and the gelatinase
was purified to high specific activity by elution of the bound material
with 4x concentrated electrophoresis sample buffer as described
in the text. Aliquots from the lysate and the gelatin-Sepharose
unbound material, the column wash, 1.0 M NaCl, and 4x electro-
phoresis sample buffer containing 8% (wt/vol) sodium dodecyl
sulfate (SDS) and 8.0 M urea as eluants were analyzed by enzymog-
raphy. Lane MGCF, monkey gingival crevicular fluid sample; lane
MPDLF, gelatin-Sepharose affinity-purified gelatinase from monkey
periodontal ligament fibroblasts. The apparent Mrs (in thousands) of
the indicated gelatinases are shown. p-gelatinase, progelatinase.
Serum albumin (A) and the electrophoresis front (f) are indicated.
molecular-mass gelatinases were totally blocked by the
divalent cation chelator EDTA and by purified human fibro-
blast tissue inhibitor of metalloproteinases (TIMP; kindly
provided by T. Cawston [Addenbrooke's Hospital, Cam-
bridge, United Kingdom]) (data not shown) but not by the
serine proteinase inhibitor PMSF. Thus, the decrease in Mr
upon APMA treatment, the divalent cation requirement for
activity, the inhibition by TIMP, and the lack of inhibition by
PMSF indicate that the 92-kDa gelatinase is a metalloprotei-
nase and is therefore likely to be MMP-9, the 92-kDa
gelatinase in the MMP family of enzymes. The identities of
the higher-M, gelatinases were not established, but they may
have also been released from polymorphonuclear leukocytes
(see below).
In an attempt to activate and reveal the presence of any
bacterial thiol proteinases in the crevicular fluid, enzymo-
gram incubations were performed either in the presence of
DTT alone or with DTT plus EDTA. However, there was no
evidence for any gelatinolytic (Fig. 2) or caseinolytic (data
not shown) activities under these incubation conditions,
indicating that these bacterial proteinases either were not
present in the crevicular fluid or, unlike the 72- and 92-kDa
gelatinases, had become irreversibly inactivated either in the
crevicular fluid or during electrophoresis.
Since human gingival fibroblast gelatinase has an Mr of
72,000 (under reducing conditions), it was unlikely that
unstimulated gingival or periodontal ligament fibroblasts
were the source of the 92-kDa gelatinase. However, to
ensure that the 92-kDa gelatinase was not normally derived
from fibroblasts in the monkey, monkey periodontal liga-
ment fibroblasts (24) were grown to confluence and made
quiescent by serum deprivation for 24 h, and the conditioned
medium was collected after another 24 h. Gelatinase was
affinity purified by gelatin-Sepharose chromatography as
described before (33, 35). Like human gingival fibroblasts,
monkey periodontal ligament fibroblasts produced 72-kDa
gelatinase (Fig. 3, lane MPDLF), some of which was acti-
vated during purification, as evidenced by the generation of
a lower-molecular-mass 64-kDa gelatinase.
Consistent with previous reports that polymorphonuclear
NOTES 4689
leukocytes are present in high numbers in periodontitis, the
gingival tissues were found to be heavily infiltrated by
polymorphonuclear leukocytes after tooth ligation (27a).
Polymorphonuclear leukocytes are known sources of pro-
teolytic enzymes (13, 27, 28, 42, 47), and polymorphonuclear
leukocyte-derived ,B-glucuronidase, lactate dehydrogenase,
(19, 20). Therefore, to determine whether polymorphonu-
clear leukocytes were a possible source of the 92-kDa
gelatinase, monkey polymorphonuclear leukocyte gelatinase
was affinity purified by gelatin-Sepharose chromatography.
Polymorphonuclear leukocytes were prepared from heparin-
ized whole-blood samples from each monkey by dextran
sedimentation (1) followed by Ficoll-Hypaque density gradi-
ent centrifugation (8) and then suspended in phosphate-
buffered saline and adjusted to 106 cells per ml. The poly-
morphonuclear leukocyte preparations were lysed by
ultrasonication in distilled water, and the lysates were clar-
ified by centrifugation (13,000 x g, 10 min) before being
loaded as either 1- or 2-ml samples onto 500-,ul minicolumns
of gelatin-Sepharose 4B (33, 35). The unbound material was
collected, and each column was washed with 2 ml of assay
buffer. Gelatin-Sepharose-bound material was then collected
by elution, first with 250 ,ul of 1.0 M NaCl in assay buffer and
then with 4 x concentrated electrophoresis sample buffer
(33, 35). As shown in Fig. 3, polymorphonuclear leukocyte
gelatinase present in the electrophoresis sample buffer eluant
electrophoresed with an apparent Mr of 92,000 and comi-
grated with the major gelatinase present in the gingival
crevicular fluid samples. In addition, minor polymorphonu-
clear leukocyte gelatinases were present at 116 kDa and at a
high, undetermined molecular mass, similar to the minor
gelatinolytic species that were variably observed in the
gingival crevicular fluid samples (Fig. 1). This indicated that
both the 92-kDa and the high-molecular-mass gelatinases
present in the gingival crevicular fluid were not of fibroblast
or osteoblast origin and may have been derived from poly-
morphonuclear leukocytes. In particular, the presence of the
116-kDa gelatinase, a characteristic of polymorphonuclear
leukocyte gelatinase activities, in the crevicular fluid sam-
ples was demonstrated more often than not (Fig. 1), provid-
ing further evidence for a polymorphonuclear leukocyte
origin for the 92-kDa gelatinase. Moreover, the occasional
presence of small amounts of the 72-kDa gelatinase (electro-
phoresing at 66 kDa under nonreducing conditions) further
indicates the minor contribution of fibroblastic MMPs in this
exudate.
Although comigration of proteins by electrophoresis does
not confer identity, additional evidence for a polymorpho-
nuclear leukocyte origin for the collagenolytic enzymes
present in gingival crevicular fluid was derived from exper-
iments using antibodies known to block the activity of
fibroblast collagenase but not that of leukocyte collagenase
(3). Procollagenase in the gingival crevicular fluid samples
was activated with 1.0 mM APMA for 30 min at 22°C (32)
before the addition of either rabbit anti-human fibroblast
collagenase antibody at various concentrations from a 1/2 to
a 1/200 dilution (antiserum was kindly provided by H.
Birkedal-Hansen, Department of Oral Biology, University of
Alabama, Birmingham) or nonimmune serum antibody, both
purified on protein A-Sepharose. After incubation at 22°C for
45 min, the collagenase assay was initiated by the addition of
2,500 dpm (-7 ng) of metabolically labeled [14C]glycine type
I collagen (32). After an 18-h incubation at 22°C, the assay
reaction products were resolved by electrophoresis on 7.5%
polyacrylamide gels (32). For positive controls, human gin-
4690 NOTES
tD
t&<
e sz4 ruC < Cw r~~~~~~~~60
(IA chains
FIG. 4. Effect of neutralizing anti-human fibroblast collagenase
antibody on gingival crevicular fluid collagenase activity. Monkey
gingival crevicular fluid (MGCF) was assayed for collagenase activ-
ity after APMA activation and the addition of either-buffer, nonim-
mune control antibody (Co Ab), or anti-human fibroblast collage-
nase antibody (Cs Ab) (1/2 and 1/200 dilutions shown) as described
in the text. After the assay, the reaction products were resolved by
electrophoresis on 7.5% polyacrylamide gels, and the [14C]glycine-
labeled al- and a2-collagen chains and the collagenase-generated
3/4-collagen (tA) bands were visualized by fluorography after expo-
sure to Kodak SB5 film at -70°C. For controls, purified human
gingival fibroblast (HGF), monkey periodontal ligament fibroblast
(MPLF), and porcine gingival explant (PGE) collagenase were used.
The collagenase inhibitor TIMP was also used as a control for
collagenase inhibition. Lane S, type I collagen substrate control
incubated in the absence of collagenase-containing samples.
gival fibroblast and monkey periodontal ligament fibroblast
collagenases were partially purified from 24-h conditioned
cell culture medium on heparin-Sepharose CL-6B (Pharma-
cia LKB Biotechnology Inc.) as described before (33, 35)
and reacted with the anti-human collagenase antibodies.
Highly purified porcine gingival explant collagenase (39) was
also used as a control. Purified human fibroblast TIMP was
used as a positive control for collagenase inhibition. The
occasional slight decrease in total (active plus latent) colla-
genase activity in crevicular fluid following APMA activa-
tion that we have observed previously (9, 17, 21) was not
specifically investigated further here because of the diffi-
culties in interpreting quantitative data from functional as-
says of samples containing a mixture of enzymes and inhib-
itors. Whereas the anti-human fibroblast collagenase
antibody totally inhibited human gingival fibroblast collage-
nase activity, it had no effect on the collagenase activity
present in the crevicular fluid samples at concentrations as
high as a 1/2 dilution of the original volume of antiserum
(Fig. 4). That this was not due to species differences in
antibody reactivity was shown by the inhibition of monkey
periodontal ligament fibroblast collagenase activity. Thus,
together with the absence of detectable bacterial collagena-
ses, the lack of inhibition of monkey gingival crevicular fluid
collagenase activity by the anti-human fibroblast collagenase
antibody provides further evidence that polymorphonuclear
leukocytes are the major source of the collagenolytic protei-
nases in the crevicular fluid.
Based on substrate specificity (14) and activation charac-
teristics (9, 42), indirect evidence for a polymorphonuclear
leukocyte source for gingival crevicular fluid collagenase has
accumulated. However, substrate specificity and enzyme
activation analyses of samples containing a mixture of en-
zymes can be difficult to interpret, and the relative propor-
tions of the constituent proteinases cannot be quantitated.
The presence of TIMP and oa2-macroglobulin in crevicular
fluid samples further complicates the interpretation of these
data, since the collagenase inhibitors complex with the
activated MMPs and only the net levels of activated colla-
INFECT. IMMUN.
genase are then detectable (35). In addition, depending on
the method of activation, different degrees of autodegrada-
cha in
tion of the activated MMPs may be observed, adding further
variability to the data. Thus, while enzyme activation and
substrate specificity analyses are useful, they only provide
indirect evidence for collagenase typing. Until blocking
antibodies to leukocyte collagenase become generally avail-
able, the use of antibodies to fibroblast collagenase also
provides only indirect, though compelling, evidence for a
polymorphonuclear leukocyte origin for crevicular fluid col-
lagenase.
Other than the minor amounts of 72-kDa gelatinase that
were occasionally present in the gingival crevicular fluid
samples, connective tissue cell-derived MMPs could not be
found in the crevicular fluid. However, this does not pre-
clude their involvement in the pathogenesis of periodontitis.
Indeed, interstitial collagenase has been identified in in-
flamed gingiva by immunolocalization (49), and on the basis
of activation characteristics, latent interstitial collagenase
was found to predominate over leukocyte collagenase at
sites of treated periodontitis (9). It is conceivable that
activated collagenase and other MMPs at the focus of
inflammation are eliminated by autodegradation (3), by re-
maining bound to insoluble collagen (34, 45), by complexing
with inhibitors, or by being cleared through the lymphatics
rather than by being cleared into the crevicular fluid.
Overall, the data presented here strongly implicate poly-
morphonuclear leukocytes as the major cellular source of
collagenolytic proteinases in the gingival crevicular fluid
during episodes of inflammation associated with connective
tissue destruction. Moreover, until now, the presence, iden-
tity, and source of gelatinases in gingival crevicular fluid was
unknown. The function of polymorphonuclear leukocytes in
the periodontium is generally considered to be one of pro-
tection. Indeed, congenital or acquired defects in polymor-
phonuclear function are often associated with rapidly de-
structive forms of periodontitis (46). Polymorphonuclear
leukocytes leave the gingival vascular plexus and migrate
through the tissue to the gingival sulcus, where they prevent
microbial invasion of the periodontium by phagocytosis and
by the release of lysosomal enzymes. However, since poly-
morphonuclear leukocyte degranulation occurs both in the
periodontal sulcus and in the tissues, the functional signifi-
cance of these proteinases in the crevicular fluid is unclear.
Nonetheless, the proportions of active gelatinase (84 kDa)
and progelatinase (92 kDa) were found to vary with time
(Fig. 1), indicating that MMP activation may reflect the
degradative process. Indeed, our previous work has re-
vealed a positive association between the presence of active
collagenase and periodontal attachment loss (22) and an
increased severity of disease (9, 17, 32, 34). Moreover, after
the resolution of periodontal inflammation, the levels of
active collagenase in the gingival crevicular fluid decrease (9,
11, 21, 22). Thus, analysis of the proteinases that may be
responsible for the destruction of the major structural pro-
teins in tissues provides a rational approach toward under-
standing the underlying mechanisms of connective tissue
destruction during inflammation.
C.M.O. is supported by a Medical Research Council of Canada
Centennial Fellowship. C.A.G.M. is supported by a Career Scientist
Award from the Ontario Ministry of Health. This work was sup-
ported by grants from the Medical Research Council of Canada and
the Ontario Ministry of Health.
VOL. 59, 1991
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