Journal of Dental Research http://jdr.sagepub.com/

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 The online version of this article can be found at: http://jdr.sagepub.com/content/71/2/359

Published by: http://www.sagepublications.com

On behalf of: International and American Associations for Dental Research

Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/71/2/359.refs.html

>> Version of Record - Feb 1, 1992 What is This?

Downloaded from jdr.sagepub.com at SYRACUSE UNIV LIBRARY on November 26, 2013 For personal use only. No other uses without permission.

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

Downloaded from jdr.sagepub.com at SYRACUSE UNIV LIBRARY on November 26, 2013 For personal use only. No other uses without permission.

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 Downloaded from jdr.sagepub.com at SYRACUSE UNIV LIBRARY on November 26, 2013 For personal use only. No other uses without permission.

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

Downloaded from jdr.sagepub.com at SYRACUSE UNIV LIBRARY on November 26, 2013 For personal use only. No other uses without permission.

.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\

Downloaded from jdr.sagepub.com at SYRACUSE UNIV LIBRARY on November 26, 2013 For personal use only. No other uses without permission.

ELASTASE AND INHIBITORS IN HUMAN GINGIVAL FLUID

Vol. 71 No. 2

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. REFERENCES

ASMAN, B.; BERGSTROM, K.; and SODER, P.O. (1981): Ratio of Alpha-1antitrypsin to Transferrin in Gingival Fluid and in Blood from Patients with Periodontal Disease, Scand JDent Res 89:407-411. ATTSTROM, R. (1975): The Roles of Gingival Epithelium and Phagocytosing Leukocytes in Gingival Defense, J Clin Periodontol 2:25-32. BAGGIOLINI, M.; BRETZ, U.; DEWALD, B.; and FEIGENSON, M.E. (1978): The Polymorphonuclear Leukocyte,AgentsActions 8:3-10. BAICI, A. (1986): Hysteretic Enzyme Response Induced by InhibitoryAntibodies against Human Leukocyte Elastase, Biol Chem Hoppe-Seyler 367:245258. BAICI, A. (1990): Interaction of Human Leukocyte Elastase with Soluble and Insoluble Protein Substrates. A Practical Kinetic Approach, Biochim BiophysActa 1040:355-364. BAUGH, R.J. and TRAVIS, J. (1976): Human Leucocyte Granule Elastase: Rapid Isolation and Characterization, Biochemistry 15:836-841. BRIGGAMAN, R.A.; SCHECHTER, N.M.; FRAKI, J.; and LAZARUS, G.S. (1984): Degradation of the Epidermal-dermal Junction by Proteolytic Enzymes from Human Skin and Human Polymorphonuclear Leukocytes, JExp Med 160:1027-1042. BRILL, N. and BRONNESTAM, R. (1960): Immuno-electrophoretic Study of Tissue Fluid from Gingival Pockets,Acta Odontol Scand 18:95-100. CASTILLO, M.J.; NAKAJIMA, K.; ZIMMERMANN, M.; and POWERS, J.C. (1979): Sensitive Substrates for Human Leukocyte and Porcine Pancreatic Elastase: A Study of the Merits of Various Chromophoric and Fluorogenic Leaving Groups in Assays for Serine Proteases,AnalBiochem 99:53-64. CERGNEUX, M.; ANDERSEN, E.; and CIMASONI, G. (1982): In vitro Breakdown of Gingival Tissue by Elastase from Human Polymorphonuclear Leukocytes. An Electron Microscopic Study, J Periodont Res 17:169-182. CIMASONI, G. and GIANNOPOULOU, C. (1988): Can Crevicular Fluid Component Analysis Assist in Diagnosis and Monitoring Periodontal Breakdown? In: Periodontology Today, B. Guggenheim, Ed., Basel: Karger, pp.260-270. CONDACCI, I.; CIMASONI, G.; and AHMAD-ZADEH, C. (1982): Alpha-2macroglobulin in Sulci from Healthy and Inflamed Gingivae, Infect Immun 36:66-71. CONDACCI, I.; CIMASONI, G.; REY, M.; and BAEHNI, P. (1988): In vitro Synthesis of a2-macroglobulin by Human Gingival Fibroblasts, Arch Oral Biol 33:407-412. GADEK, J.E.; FELLS, G.A.; ZIMMERMAN, R.L.; RENNARD, S.I.; and CRYSTAL, R.G. (1981): Antielastases of the Human Alveolar Structures: Implications for the Protease-antiprotease Theory of Emphysema, J Clin Invest 68:889-898. GIANNOPOULOU, C.; DI FELICE, R.; ANDERSEN, E.; and CIMASONI, G. (1990): Synthesis of a2-macroglobulin in Human Gingiva: a Study of the Concentration of Macroglobulin and Albumin in Gingival Fluid and Serum,Arch Oral Biot 35:13-16. KLEESIEK, K.; NEUMANN, S.; and GREILING, H. (1982): Determination of the Elastase al-proteinase Inhibitor Complex, Elastase Activity and Proteinase Inhibitors in the Synovial Fluid, Fresenius Z Anal Chem 311:434-435.

KOWASHI, Y.; JACCARD, F.; and CIMASONI, G. (1979): Increase of Free Collagenase and Neutral Protease Activities in the Gingival Crevice

363

during Experimental Gingivitis in Man,Arch Oral Biol 24:645-650. KRYSHTALSKYJ, E.; SODEK, J.; and FERRIER, J.M. (1986): Correlation of Collagenolytic Enzymes and Inhibitors in Gingival Crevicular Fluid with Clinical and Microscopic Changes in Experimental Periodontitis in the Dog,Arch OralBiol 31:21-31. LOE, H. (1967): The Gingival Index, the Plaque Index and the Retention Index Systems, JPeriodontol 38:610-616. MEYLE,J.; HELLER, W.; andFUHRER, G. (1989): Simultaneous Determination ofFree and Complexed Elastase in Gingival Crevicular Fluid (GCF), J Dent Res 68:631,Abst. No. 178. MUNCK-PETERSEN, C.; VESTERGARD-POVLSEN, J.; and INGERSLEV, J. (1985): Enzyme-linked Immunosorbent Assay (ELISA) for the Measurement of Small Quantities of a2-macroglobulin, Scand J Clin Lab Invest 45:735-740. NEUMANN, S.; GUNZER, G.; HENNRICH, N.; and LANG, H. (1984): PMNelastase Assay: Enzyme Immunoassay for Human Polymorphonuclear Elastase Complexed with ni-proteinase Inhibitor, J Clin Chem Clin Biochem 22:693-697. OHLSSON, K. and OLSSON, I. (1974a): The Neutral Proteases of Human Granulocytes. Isolation and Partial Characterization of Granulocyte Elastases, EurJBiochem 42:519-527. OHLSSON, K. and OLSSON, I. (1974b): Neutral Proteases of Human Granulocytes. III. Interaction between Human Granulocyte Elastase and Plasma Protease Inhibitors, Scand J Clin Lab Invest 34:349-355. OHLSSON, K.; OLSSON, I.; and TYNELIUS-BRATTHALL, G. (1973): Neutrophil Leukocyte Collagenase, Elastase and Serum Protease Inhibitors in Human Gingival CrevicesActa Odontol Scand 31:51-59. SALVESEN, G.; VIRCA, G.D.; and TRAVIS, J. (1983): Interaction of Alpha-2macroglobulin with Neutrophil and Plasma Proteinases,Ann NYAcad Sci 421:316-326. SCHENKEIN, H.A. and GENCO, R.J. (1977): Gingival Fluid and Serum in Periodontal Diseases. I. Quantitative Studyoflmmunoglobulins, Complement Components, and other Plasma Proteins, JPeriodontol 48:772-777. SENGUPTA, S.; LAMSTER, I.B.; KHOCHT, A.; DUFFY, T.A.; and GORDON, J.M. (1988): The Effect ofTreatment on IgG, IgA, IgM and a2-macroglobulin in Gingival Crevicular Fluid from Patients with Chronic Adult Periodontitis,Arch OralBiol 33:425-431. SKALERIC, U.; ZAJSEK, P.; CVETKO, E.; LAH, T.; and BABNIK, J. (1986): Alpha-2-macroglobulin in Gingival Fluid: Correlation with Alveolar Bone Loss in Periodontal Disease, J Clin Periodontol 13:833-836. SUEDA, T.; BANG, J.; and CIMASONI, G. (1969): Collection of Gingival Fluid for Quantitative Analysis, JDentRes 48:159. TESTA, L.D.; KUCICH, U.; and LALLY, E.T. (1986): Immunologic Measurement of Gingival Crevicular Fluid (GCF) Human PMN Elastase (hPMNe) Levels,J DentRes 65:183,Abst. No. 119. TOLLEFSEN, T. and SALTVEDT, E. (1980): Comparative Analysis of Gingival Fluid and Plasma by Crossed Immunoelectrophoresis, JPeriodont Res 15:96-106.

TRAVIS, J.; BAUGH, R.; GILES, P.J.; JOHNSON, D.; BOWEN, J.; and REILLY, C.F. (1978): Human Leukocyte Elastase and Cathepsin G: Isolation, Characterization and Interaction with Plasma Proteinase Inhibitors. In: Neutral Proteases of Human Polymorphonuclear Leukocytes, K. Havemann and A. Janoff, Eds., Baltimore: Urban and Schwarzenberg, pp. 118-128. VIRCA, G.D. and SCHNEBLI, H.P. (1984): The Elastase/ol-proteinase Inhibitor Balance in the Lung, Schweiz Med Wschr 114:895-898. VOLLER, A.; BIDWELL, D.; and BARTLETT, A. (1976): Microplate Enzyme Immunoassays for the Immunodiagnosis ofVirus Infections. In: Manual of Clinical Immunology, N.R. Rose and H. Friedman, Eds., Washington, DC: American Societyfor Microbiology, pp.506-512. WEISS, S.J. (1989): Tissue Destruction by Neutrophils, New Engl J Med

320:365-376.

WEWERS, M.D.; HERZYK, D.J.; and GADEK, J.E. (1988): Alveolar Fluid Neutrophil Elastase Activity in the Adult Respiratory Distress Syndrome is Complexed to Alpha-2-macroglobulin, J Clin Invest 82:1260-1267.

Downloaded from jdr.sagepub.com at SYRACUSE UNIV LIBRARY on November 26, 2013 For personal use only. No other uses without permission.

Neutrophil elastase and its inhibitors in human gingival crevicular fluid during experimental gingivitis.

The relative concentrations and absolute amounts of neutrophil elastase and its two inhibitors, alpha 2-macroglobulin (alpha 2-M) and alpha 1-antitryp...
679KB Sizes 0 Downloads 0 Views