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Neutrophil Elastase and its Inhibitors in Human Gingival Crevicular Fluid during Experimental
Gingivitis
C. Giannopoulou, E. Andersen, C. Demeurisse and G. Cimasoni
J DENT RES 1992 71: 359
DOI: 10.1177/00220345920710020301
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Neutrophil Elastase and its Inhibitors in Human Gingival Crevicular Fluid
during Experimental Gingivitis
C. GIANNOPOULOU, E. ANDERSEN, C. DEMEURISSE, and G. CIMASONI
Division of Physiopathology and Periodontology, School of Dentistry, Medical Faculty, University of Geneva, 19, rue
Barthelemy Menn, 1211 Genkve 4, Switzerland
The relative concentrations and absolute amounts of neutrophil
elastase and its two inhibitors, a2-macroglobulin (a2-M) and alantitrypsin (al-AT), were determined in gingival crevicular fluid
(GCF) collected from six dental students who refrained from brushing the upper left or right quadrant during three weeks. Plaque and
gingival indices and flow ofGCF were measured before, during, and
after the three weeks of no brushing. Functional elastase, representing the enzyme complexed with a2-M, was measured by use of
a low-molecular-weight fluorogenic substrate. Elastolytic activity
in GCF was also assayed by use of elastin as substrate. Antigenic
elastase, representing the enzyme complexed with al-AT, as well as
the inhibitors a2-M and al-AT were measured by ELISA. After
three weeks of plaque accumulation, the concentrations of both
functional and antigenic elastase increased by a factor of about 3,
whereas the concentrations of the inhibitors increased in a much
higher proportion. No free elastase could be detected in GCF, as
evidenced by the Sephadex G-75 elution profile of GCF, by the
negative results obtained when elastin was used as substrate, and
by the demonstration that pure enzyme kept its activity against the
low-molecular-weight substrate after being saturated by a2-M.
J Dent Res 71(2):359-363, February, 1992
Introduction.
Leukocyte elastase is one of the major enzymes of the azurophilic
granules of human neutrophils, and in inflammation its release
could contribute to tissue damage (Baggiolini et al., 1978; Virca and
Schnebli, 1984). Biopsies of skin experimentally treated with purified elastase showed gradual degradation of the epidermal-dermal
junction (Briggaman et al., 1984). Similarly, biopsies of human
gingiva treated with low concentrations of the enzyme showed
enlargement of the epithelial intercellular spaces, interruptions of
the basement lamina, and loss of collagen (Cergneux et al., 1982).
The presence of elastase in crevicular material or gingival
crevicular fluid (GCF) has been demonstrated in several laboratories using different techniques. Ohlsson et al. (1973), using an
immunochemical method, showed that crevicular material from
chronically inflamed gingivae contains about seven times more
enzyme than material from healthy sulci. Kowashi et al. (1979)
followed the activity of elastase in the gingival washings from eight
human volunteers during an experimental gingivitis and confirmed
that the enzyme activity increased significantly at the peak of the
inflammatory reaction. Enzyme-linked immunosorbent assay
(ELISA) was used by Testa et al. (1986) for determination of elastase
in the GCF of four groups of patients with increasing degrees of
gingival disease. The levels of this antigenic elastase were found to
increase in the GCF of patients with deeper pockets. Finally, Meyle
et al. (1989), using a commercially available ELISA kit, found a
higher concentration of free elastase in GCF from patients with
periodontitis as compared with that in GCF from healthy volunteers.
The activity of proteases in the tissues is modulated by the
presence of inhibitors either produced locally or circulating in
Received for publication August 4, 1990
Accepted for publication September 18, 1991
plasma (Travis et al., 1978). The presence of alpha-1-antitrypsin
(al-AT) and especially of alpha-2-macroglobulin (a2-M) has been
shown in GCF or gingival washings by a number of investigators
using different techniques (Brill and Bronnestam, 1960; Ohlsson et
al., 1973; Schenkein and Genco, 1977; Tollefsen and Saltvedt, 1980;
Asman et al., 1981; Condacci et al., 1982; Skaleric et al., 1986;
Sengupta et al., 1988).
Techniques are now available which allow functional elastase,
antigenic elastase, and the inhibitors a2-M and al-AT to be measured in the same microsample. Functional elastase can be measured by use of low-molecular-weight substrates; these are known
to be attacked not only by free molecules of enzyme but also by
elastase trapped by a2-M (Salvesen et al., 1983; Wewers et al.,
1988). Antigenic elastase can be determined by ELISA: When antielastase is used as primary antibody and anti-al-AT as second
antibody, the complex elastase-al-AT will be measured (Neumann
et al., 1984). However, when anti-elastase is used as second antibody, the assay will measure both free elastase and the complex
elastase-al-AT. Finally, the inhibitors al-AT and a2-M, both free
and bound to proteases, can also be measured by ELISA (Gadek et
al., 1981; Munck-Petersen et al., 1985).
In the present investigation, the presence of functional and
antigenic elastase and its inhibitors in human GCF was followed
longitudinally during an experimental gingivitis.
Materials and methods.
Experimental gingivitis. -An experimental gingivitis was obtained
in six dental students, from 23 to 26 years old. First, the students
underwent a thorough prophylaxis and were recommended to maintain perfect oral hygiene. They were examined each day until the
plaque and inflammation indices reached the value of 0. The
volunteers were then observed for eight days before the "no brushing" period (day 0) was initiated. They were then asked to stop oral
hygiene on the left or right upper quadrant for 20 days, and were
followed for 12 supplemental days. The Gingival Index (Loe, 1967)
was used to measure the degree of inflammation and the Plaque
Index (Loe, 1967) for the amount of plaque. The two indices were
taken on the mesial aspects ofthree teeth (canine, premolar, or first
molar) in the selected upper quadrant for each volunteer where no
restoration was present. Gingival crevicular fluid (GCF) was
collected with a recently standardized intracrevicular technique
from the same three sites (Cimasoni and Giannopoulou, 1988). The
region was first isolated with cotton rolls and dried with air for
several seconds. After two min, the tips of Durapore strips (0.22-pm
pore, 2 x 5 mm dimension; Millipore, Bedford, MA) were inserted
into the crevices until mild resistance was felt and left in place for
30 s. The strips were weighed before and immediately after the
collection, within a sealed microcentrifuge plastic tube. The difference between the two weighings gave the volume of fluid collected,
assuming a specific gravity of approximately 1. Rubber gloves were
used throughout the procedure to avoid sample contamination.
Evaporation of GCF was kept to a minimum by limiting to a few
seconds the time during which the tube remained open (Cimasoni
and Giannopoulou, 1988). All samples of fluid were obtained before
the determination of the plaque and gingival indices in the same
sites, in order to avoid the effects of gingival irritation. The Plaque
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359
360
GIANNOPOULO U et al.
J Dent Res
Elastase (antigenic)
Elastase Activity
800
February 1992
D Concentration
1200
D Concentration
1000
L3mTotal
(ng)
800
800
(Eug/mL)
(1(pg/mL)
0 Total
us (ng)
600
600
I
-A
m 400
4-
-J
400 cC)
E 600
600
400
400
c
200
0I
200
T
~ILL
I
200
J,
lil
.....
-.
-8
-3
-1
.
_.-
ri ik rm
I
0 +20 +22 +26 +32
DAYS
Fig. 1-Elastase activity (functional) in crevicular fluid during experimental gingivitis. Each column represents the average concentration and
total amount from 18 sites from six subjects and the vertical bars the
standard deviations.
0
Ad
-8
aY1 U.S*,
-3
-1
r..=
0
P-;,
200
h ;
U1-.
F.I.-
,
+20 +22 +26 i32
DAYS
Fig. 2-Average concentrations and total amounts (± SD) of antigenic
elastase during the period of experimental gingivitis.
as control. A linear response was obtained over a range from 0.09
to 1.42 ig/mL.
To test whether the presence of a2-M modified the activity of
Index, the Gingival Index, and the flow of GCF were measured at
days -8, -3, and -1. Plaque and Gingival Indices were again elastase, as determined by our low-molecular-weight substrate,
determined at days 0, +7, +14, and +20. Because of the presence of identical amounts of purified enzyme were tested with this subincreasing amounts of plaque, GCF was collected only at days 0 and strate in a preliminary assay, both in the absence and in the
+20. At day +20, the volunteers resumed their normal hygiene presence of purified a2-M (DAKO, 2600, Glostrup, Denmark).
For determination ofthe antigenic elastase, either free or bound
habits, and the two indices as well as the flow of GCF were again
to al-AT, the following procedure was used: The wells of the
measured at days +22, +26, and +32.
Determination of enzyme and inhibitors.-Antigenic elastase immunoplates were coated with 200 pL of rabbit antisera (1 p/mL)
and the inhibitors a2-M and al-AT were measured in the samples against human elastase, and the plates were then stored at 40C for at
of fluid by ELISA, and the activity of elastase (functional) was least 15 h before use. They were then washed three times with 0.1
analyzed by the use of a specific low-molecular-weight fluorogenic mol/L PBS containing 0.05% Tween 20 (PBS-T). A100-pL quantity of
a dilution of pure elastase (purified in our laboratory from human
substrate.
After the samples were collected and weighed, phosphate-buff- PMNs, at a concentration of 0.7 g/L) was used for the standard curve
ered saline (PBS), pH 7.2, was added to each microtube to obtain a (dilution range, 1:2000 to 1:128,000). Optimum dilutions ofGCF were
1:500 dilution of the GCF. Each tube was then shaken on a vortex found to be of 1:200,000. After a two-hour incubation, 200 pL ofthe
mixer for one min and centrifuged for seven min at 1200 g. After anti-elastase, conjugated to alkaline phosphatase and diluted 1:4000,
centrifugation, the supernatant was divided into two fractions: one was applied. The conjugate was prepared as described byVolleret al.
for determination of the enzyme activity, and the other for the (1976): It consisted of the antibody, labeled with alkaline phosdetermination-ofthe amounts ofantigenic elastase and ofa2-M and phatase, and could react with any antigen already captured by the
al-AT. The samples were frozen at -20'C until the day of analysis. antibody at the well surface. Finally, 200 jib of the substrate pFor the determination of elastase activity (Castillo et al., 1979), nitrophenylphosphate (1 mg/mL), diluted in 10% diethanolamine
either free or bound to the inhibitor a2-M, the fluorogenic substrate buffer, pH 9.8, was added to each well and the reaction stopped with
Meo-Suc-Ala-Ala-Pro-Val/7-amino-4-methylcoumarin(MW627.69) 50 jL of a 3 mol/L solution of NaOH.
The two inhibitors were assayed by the following procedure: The
(Bachem, Bubendorf, Switzerland) was used. The enzyme assay
was conducted at 250C with 0.1 mmolL substrate in 50 mmol/L wells ofthe immunoplates were coated with 200 pL of commerciallyphosphate buffer, pH 7.4, containing 0.05% Triton X-100 and 0.5 available rabbit antibodies (1 pg/mL) against human a2-M and almol/L NaCl (Baici, 1986). One hundred pL of the substrate solution AT (DAKO, 2600, Glostrup, Denmark) and stored at 40C for at least
was pre-incubated for five min, and 20 pL of a sample of GCF 15 h before use. Afterthe washing procedure with PBS-T, the serum
(dilution 1:500) was added. After six h of incubation, the reaction of a healthy person was used for the standard curve. This serum had
was stopped with 5 jL of a selective enzyme inhibitor, a concentration of 1.25 g/L for both a2-M and a 1-AT and was applied
phenylmethylsulfonylfluoride (PMSF), at a final concentration of in the following dilution range: 1:18,000 to 1:1,152,000 for a2-M and
1.9 mmol/L (Sigma, St. Louis, MO). Fluorescence ofthe 7-amino-4- 1:12,500 to 1:1,250,000 for a 1-AT. Optimum dilutions for GCF were
methylcoumarin (H-AMC) was monitored by means of a found to be of 1:50,000 for a2-M and of 1:100,000 for al-AT. After
spectrofluorimeter SFM 23 (Kontron Inc., Princeton, NJ). Excita- a two-hour incubation, 200 pL of antiserum labeled with alkaline
tion and emission wavelengths were 383 and 460 nm, respectively. phosphatase (dilutions: 1:2000 for o2-M and 1:1000 for a1-AT) was
All the readings were performed in a Hellma ultra-micro-cell cu- applied. This conjugate was also prepared according to the procevette (type 105.251-QS). The standard curve was established with dure outlinedbyVolleretal. (1976). Finally, 200 b ofthe substrate
H-AMC (Bachem, Bubendorf, Switzerland), and a linear response p-nitrophenylphosphate (1 mg/mL) was added to each well and the
was obtained over the concentration range of 0.35-140 pmolfL. A reaction stopped with 50 pL of the 3 mol/L NaOH.
All the results will be expressed both as "concentration", indesecond standard curve of pure elastase, purified in our laboratory
(Baugh and Travis, 1976) and ofknown concentration, was also used pendent of fluid flow rate, and "amount", i.e., the total content in a
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1200
1200
Concentration
(pg/mL)
Total
1000
;.IF- (ng)
1000
800
800
800
600
600
-j
0)
1
600
c
400
400
400
200
200
200
I
L'.J
I
I,
-8
Easx,_.In|arm -sfi |-v
aIAT
*2-M
0
361
ELASTASE AND INHIBITORS IN HUMAN GINGIVAL FLUID
Vol. 71 No. 2
-3
A
M-3
-1
0
U
r--~~~~~~~~~~~~~~~~~.~
+20 +22 +26 +32
DAYS
0o
~'IJJLKL
fi
-8
-3
-1
Ih.it
D Concentration
(jig/mL)
E] Total
(ng)
800
600
c
400
200
n- +20 +22 +26 +32
0
DAYS
Fig. 3-Alpha-2-macroglobulin (concentration and total) in crevicular
fluid before, during, and after the period of plaque accumulation.
Fig. 4-Concentrations and total amounts of alpha-1-antitrypsin in the
fluid before, during, and after the period of plaque accumulation.
given sample of GCF.
Chromatography ofgingival crevicular fluid.-To characterize
the molecular size distribution of functional and antigenic elastase,
four samples of GCF, collected by capillaries from five patients
(Sueda et al., 1969), were applied to a Sephadex G-75 column having
a length of 80 cm and a diameter of 1.6 cm. A first sample of 35 pL
pooled from two patients was diluted in 2 mL ofPBS and applied to
the column. Three further samples of 2 pL of GCF collected from
three different patients were also applied. The samples were eluted
in PBS at 4VC, and 1.1-mL fractions were collected for determination of functional elastase, antigenic elastase, and the inhibitors, as
described above. In separate experiments, samples of commercially
available a2-M (0.85 mg dissolved in 2 mL of PBS), of commercially
available al-AT (0.7 mg in 2 mL of PBS), and of neutrophil elastase
(37 pg diluted in 2 mL of PBS) were applied to the same column, and
their presence and position in the eluate were determined by
ELISA, as explained above.
Elastin assay.-The ability of GCF to solubilize elastin was
checked by means of the fluorimetric technique of Baici (1990).
Briefly, samples of from 1 to 3 jiL of GCF or serum and 2.0 mg of
elastin from bovine neck ligament (Sigma) were incubated for four
h at 370C in 430 pL of Na+/K+ phosphate buffer, 50 mmol/L. The
reaction was stopped by addition of 70 pL of 36% trichloroacetic acid.
Aftercentrifugationfor sixmin at 600g, a 0. 1-mL portion ofthe clear
supernatant was added to 3.0 mL of 0.2 mol/L sodium borate buffer,
pH 8.5. Finally, 1.0 mL of a solution of fluorescamine (Sigma) (15
mg/100 mL acetone) was added, and the fluorescence of the liberated peptides was monitored in a spectrofluorimeter. Excitation
and emission wavelengths were 390 and 480 nm, respectively. A
standard curve was established by use of PMN elastase purified in
our laboratory (Baugh and Travis, 1976). The technique was also
tested with preparations of homogenized human blood PMNs.
determined with the low-molecular-weight substrate increased
from 200 (± 152) pg/mL at day 0 to 428 (± 105) pg/mL at day +20 (p
< 0.001) and its total amount from 30 (± 34) ng to 265 (± 138) ng (p
< 0.001) (Fig. 1). Both concentration and total amount returned to
baseline values after the volunteers resumed their usual oral
hygiene.
In a preliminary assay in vitro, it was ascertained that the
activity of elastase, as determined with the low-molecular-weight
substrate, was not modified by the presence of a2-M.
Results of ELISA.-The concentration of antigenic elastase
increased from 131 (±45) pg/mL to 557 (+278) jg/mL at day +20 and
decreased afterward (Fig. 2, p < 0.001). The total amount of
antigenic elastase increased from 23 (± 13) ng at day 0 to 322 (± 183)
ng at day +20 (p < 0.001, Fig. 2). The concentrations and total
amounts of a2-M and al-AT also increased significantly during the
inflammatory reaction and decreased afterwards (Figs. 3 and 4).
Sephadex G-75 chromatography of gingival crevicular fluid.Fig. 5 outlines the elution profiles of the two inhibitors and of
elastase, fractionated by G-75 sieve chromatography. The profiles of
commercially-available a2-M and al-AT as well as that of pure
elastase are shown in the upper part ofthe Fig. (A), while the lower
part (B) shows the profiles offunctional and antigenic elastase and of
the two inhibitors in a sample of human GCF pooled from two
patients. As expected, the two inhibitors eluted from GCF in the
same fractions as when purified preparations of a2-M and al-AT
were used. The Fig. shows that the functional elastase of GCF coeluted with the high-molecular-mass a2-M, whereas the antigenic
elastase eluted within the peak ofthe lower-molecular-mass a 1-AT.
There was no antigenic or functional elastase detectable at molecular
masses lower than that of al-AT, where the peak of the enzyme, as
shown in A, could be expected, indicating the absence offree elastase
in GCF. Similar profiles were obtained when three samples of GCF
from three different patients were eluted through the same column.
Elastin assays.-When the activity of purified elastase against
elastin was tested, a linear response offluorescence was obtained in
the range from 0.07 pg to 0.28 pg of enzyme. Further, when the
elastolytic activity of homogenized PMNs was tested, a linear
response was also obtained in the range from 2 x 104 to 8 x 104 cells.
As expected, no elastolytic activity was found in human serum.
Indeed, the fluorescence obtained with either 1 pL, 2 jL, or 3 pL
of serum from two volunteers could be accounted for by the nonenzymatic hydrolysis of elastin and by the background fluorescence of serum itself.
Results.
Clinical findings.-As expected, the percentage of sites with no
plaque and without inflammation (degree 0) decreased during the
no-brushing period and increased rapidly thereafter, whereas the
proportion of sites with plaque or inflammation followed the reverse
trend. The flow ofGCF increased from an average baseline value of
180 nL/30 s/sample to 595 nL/30 s/sample at day +20 and returned
to pre-gingivitis levels at the end of the experiment.
Activity ofelastase.-The concentration of functional elastase as
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.0
GIANNOPOULOU et al.
362
J Dent Res
February 1992
studied. Ohlsson et al. (1973)
observed that the protease-inhibiting capacity of ai-AT was
Elastase
A. Purified
120 60
saturated in GCF from both
cE
preparations
\> healthy and inflamed gingival
a
sulci, and, more recently, Meyle
80 F
/ 40 , et al. (1989) reported, in conM
trast to the present findings,
?5
(n that GCF contained free elas40 20 C
tase in concentrations propor/
tional to the severity of gingiCAM
val inflammation. Meyle et al.
o
Z (1989) used a commerciallyavailable ELISA kit for the
determination of elastase bound
to oi-AT in a given aliquot of
GCF (Neumann et al., 1984).
1600 F
. They then saturated an identical aliquot of fluid with serum
3
and found a higher amount of
B. Gingival
300
the above complex. They con-E
crevicular
CD
' cluded that free enzyme was
fluid
C
present in GCF. In our inves800
200 2.
°°) tigation, functional elastase was
csk
I-a
determined by use of both a
substrate
low-molecular-weight
400
10
and elastin. No free activity
could be detected, as evidenced
D
by (a) the Sephadex G-75 elution profile, (b) the demonstra60 62I
tion, in vitro, thatpure enzyme,
24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62
after saturation with a2-M, kept
its activity against the low-moFractions
lecular-weight substrate, and
Fig. 5-Sephadex G-75 elution profiles. (A) Elution of purified preparaitions of A2-M (-), ni-AT (A), and
(c) the absence of elastolytic
elastase (U) as determined by ELISA. (B) Elution of functional elastase (0, c-rossed area) and antigenic elastase activity in GCF.
(A, dotted area) in a sample of human GCF, in relation to the profiles of n2'-M and an-AT in the same sample.
Since the molar combining
Fraction numbers are recorded on the abscissa. The scales for the inhibitors I(left side) and for antigenic elastase
ratio for elastase/c 1-AT is 1:1
(right) have been expanded in the upper Fig.
and that for elastase/a2-M is
2:1 (Ohlsson and Olsson, 1974b),
The GCF also did not show any elastolytic activity: Seven and considering the high molecular weight of the inhibitors, one
measurements with 1, 2, or 3 AL of fluid from three patients could taise the objection that the amounts of an-AT and a2-M at
showed a level of fluorescence which was due only to non-enzy- the peak of the inflammatory reaction were not sufficient to
matic hydrolysis of elastin and to the background fluorescence of saturate the enzyme completely, the more so that other proteases
GCF.
are found in GCF. Two factors could explain this apparent discrepancy. First, it is probable that the elastase prepared in our
was not entirely pure, and that the antibody raised
laboratory
Discussion.
against it and utilized in the ELISA determinations also recogNeutrophil elastase-both functional and antigenic-and its main nized some foreign proteins. Second, it could be possible, as with
inhibitors, (x2-M and cl-AT, were found to increase in GCF of collagenase, that part of the elastase determined by ELISA could
human volunteers during an experimental gingivitis. The in- exist in a latent form or could be bound to a different inhibitor.
Kryshtalskyj et al. (1986) have found active collagenase in dog
creases were significant when the parameters were expressed as
concentration or as total amount. As discussed by Cimasoni and GCF and have shown that the concentration of active enzyme in
Giannopoulou (1988), the increase of the amount of elastase in the fluid collected from sites with gingivitis was lower than that
GCF with inflammation is due primarily to the parallel increase of the active enzyme in the GCF from sites with periodontitis.
in the number of sulcular PMNs. Furthermore, the functions of Also, the inhibitor activities and the proportion of latent collagthese cells, such as phagocytosis (Attstrom, 1975), are stimulated enase were higher in the gingivitis sites.
Our results do not completely rule out the possibility that some
during the time of plaque accumulation, with an increased liberation of enzyme. As for the inhibitors a2-M and ni-AT, these are free elastase could be found in GCF in situations of severe
known to become more abundant during an inflammatory reac- inflammation. However, our finding-that human GCF does not
tion (Ohlsson and Olsson, 1974a). In this respect, recent results seem to contain free elastase-confirms those of Wewers et al.
from our laboratory have shown, both in vitro (Condacci et al., (1988) for alveolar fluid of patients with adult respiratory distress
1988) and in vivo (Giannopoulou et al., 1990), that a2-M can be syndrome and those of Kleesiek et al. (1982) for synovial fluid of
synthesized locally by gingival cells and that such a synthesis inflamed joints.
Elastase or other proteases might exert their deleterious
increases during inflammation.
In the gingivae, both proteases and inhibitors have frequently effects locally, immediately after being liberated from the inflambeen determined, but the balance between the two has rarely been matory cells and before being blocked by inhibitors. Recent
X2 M
I
I
_
1200
0
58\
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Vol. 71 No. 2
ELASTASE AND INHIBITORS IN HUMAN GINGIVAL FLUID
evidence has also shown that the protective role of inhibitors can
be jeopardized by the presence of oxygen radicals, known to be
produced by stimulated PMNs (Weiss, 1989).
Acknowledgments.
We thank Dr. A. Baici, Department of Rheumatology, University
Hospital, Zurich, for his helpful suggestions concerning the elastin
assays, and Mrs. H. Bosshard for typing the manuscript.
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