Vol. 59, No. 12INFECTION AND IMMUNITY, Dec. 1991, p. 4687-46920019-9567/91/124687-06$02.00/0Copyright © 1991, American Society for Microbiology

Evidence for Polymorphonuclear Leukocyte Collagenase and92-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 CouncilGroup 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 monkeyshas revealed the presence of coliagenase and a 92-kDa gelatinase that comigrated after electrophoresis with the92-kDa gelatinase released from polymorphonuclear leukocytes. Since neutralizing antibodies to fibroblastcollagenase 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 fluidfrom gingiva.

Degradation of the extracellular matrix is an importantcharacteristic of acute and chronic inflammatory lesions, butthe mechanisms of tissue degradation have not been clearlyestablished (41, 47). The characterization of the proteinasespresent in inflammatory exudates can identify the cellularsource of the enzymes and hence indicate the relativeimportance of those cells in the degradative processes oc-curring in the disease. Inflammatory diseases of connectivetissues, such as periodontitis, appear to involve connectivetissue-derived metalloproteinases, such as the interstitialcollagenase of the matrix metalloproteinase (MMP) family(10, 22, 34, 45), and a distinct collagenase, released onlyfrom polymorphonuclear leukocytes, termed leukocyte col-lagenase. In periodontitis, collagenases synthesized bypathogenic microorganisms may also contribute to collagenloss (5).

Interstitial collagenase (MMP-1) is synthesized by severalcell 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 storagegranules in polymorphonuclear leukocytes (13, 27), andalthough the enzyme cleaves the same scissile bonds incollagens as interstitial collagenase does, it differs frominterstitial collagenase in its Mr (27), substrate kinetics (14),and antigenic properties (12). The collagenolytic cascade isalso thought to involve the activity of gelatinases (41) thatcan degrade denatured collagens and native type IV colla-gen. A 72-kDa gelatinase (MMP-2) is synthesized by variousmesenchymal cells (7), including human gingival fibroblasts(35, 37) and osteoblasts (30, 32, 36), whereas a 92-kDagelatinase (MMP-9) is synthesized by polymorphonuclearleukocytes (27), alveolar macrophages (48), keratinocytes(48), and some transformed cells (48) but not by normalfibroblasts (7, 48) or osteoblasts (36). Endothelial cellsproduce 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 tumornecrosis factor (25), from macrophages (15) and lympho-cytes; these inflammatory mediators in turn induce MMPexpression in fibroblasts, macrophages, and other resident

* Corresponding author.

tissue cells. Bacterial cell surface lectins may also directlyinduce MMP expression and MMP activation in fibroblasts(33).To understand the mechanisms of tissue destruction in

periodontal inflammation, the cellular origin of proteolyticenzymes and the relative importance of host cells andbacterial cells as sources of these enzymes need to beestablished. Although some indirect evidence indicates thatpolymorphonuclear leukocytes are the source of collagenasein gingival inflammatory exudate (9, 28, 42), this has notbeen shown unequivocally. We have addressed this problemby 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, inflammationcan be induced at will, and the ensuing tissue destructionoccurs in a relatively predictable temporal manner.

Periodontitis was induced in clinically healthy gingivaaround the maxillary incisors of two 4-kg cynomolgus mon-keys (Connaught Laboratories, Toronto, Ontario, Canada)by placement of subgingival silk ligatures and withdrawal ofdaily prophylaxis for 10 weeks. This method encourageslarge increases in numbers of tooth-bound colonies of micro-organisms. Unligated mandibular incisors served as con-trols. Full descriptions of the clinical measurements andindices that were recorded weekly to monitor the loss of thegingival connective tissue attachment around these teeth andthe development of periodontitis are given in detail else-where (2). In summary, the microbial load and the amount ofinflammation reached maximum values within 1 week, gin-gival crevicular fluid flow was significantly increased at 1 and3 weeks, and there was a progressive loss of connectivetissue attachment around the experimental teeth during the10-week experiment.

Gingival crevicular fluid was collected in microcapillarytubes (sample volume, 0.66 + 0.21 ,ul [mean + standarddeviation]) (17), diluted 1/10 with assay buffer (50 mMTris-HCl, 200 mM NaCl, 5 mM CaCl2, 0.5 jig of Brij 35 perml, 0.2 jig of NaN3 per ml [pH 7.2]), and divided into fourequal aliquots, three of which were used for active and totalcollagenase and collagenase inhibitor assays as describedpreviously (2). The remaining aliquot of the crevicular fluid

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FIG. 1. Enzymogram analysis of gingival crevicular fluid. On theenzymograms, gelatinolytic proteinases in gingival crevicular fluidare revealed as clear bands against the blue-stained gelatin substratebackground. In the examples shown, samples of gingival crevicularfluid were obtained from two sites of experimental periodontitis (Aand 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 presentin the conditioned culture medium of human gingival fibroblasts waselectrophoresed nonreduced as a standard. (The 72-kDa gelatinasemigrates with an apparent Mr of 72,000 when electrophoresed underreducing conditions and with an apparent Mr of 66,000 undernonreducing conditions.) Arrow A indicates serum albumin in thecrevicular fluid. The positions of migration ofMr marker proteins (inthousands) 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% polyacrylamideseparating gels (18) containing either gelatin or casein (40p.g/ml), and enzymography was performed as describedpreviously (31). The gelatinolytic and caseinolytic protein-ases that renatured and were functionally active in the200-min assays were visualized as cleared bands against theaqua blue-stained substrate gel. For standards, the condi-tioned medium of confluent quiescent cultures of humangingival fibroblasts (Gin-i [CRL 1292]; American Type Cul-ture Collection, Rockville, Md.) was collected as a source offibroblast 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 84kDa (Fig. 1). In addition, a minor gelatinolytic doublet at 66and 59 kDa, which comigrated with the human gingivalfibroblast gelatinase, was frequently observed together withgelatinase activity in the 40-kDa region, at 116 kDa, and of avery high molecular mass. To assay for stromelysin(MMP-3) and bacterial proteinases, casein substrate gelswere used, but caseinolytic activity was never detected inany of the samples, even after prolonged assay incubationtimes (24 and 48 h; Fig. 2).To provide sufficient material for characterization of the

gingival crevicular fluid gelatinases, replicate aliquots of thediluted crevicular fluid samples, collected site specifically,were pooled. Samples were incubated with either 1.0 mMp-aminophenylmercuric acetate (APMA) or solvent at 22°Cfor 30 min. The organomercurial APMA activates pro-MMPs(43) and inhibits thiol proteinases, including the Porphory-monas gingivalis collagenase (5). Electrophoresis samplebuffer was then added, and the samples were electro-phoresed on gelatin substrate or casein substrate gels beforeincubation in assay buffer. Enzyme activity was comparedby using gels on which identical samples had been electro-phoresed but each had been incubated either in assay buffer

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FIG. 2. Characterization of gingival crevicular fluid gelatinases.Pooled gingival crevicular fluid samples (lanes G) were electro-phoresed in duplicate on substrate-polyacrylamide gels containing40 ,ug of gelatin or casein per ml as described in the text. Proteolyticactivity was assayed by enzymography with assay buffer alone(Control) or assay buffer containing either 10 mM EDTA, 10 mMDTT, 10 mM DTT plus 10 mM EDTA, 1 mM PMSF, or 5 mM NEMas indicated. To activate any progelatinases present in the gingivalcrevicular fluid, the samples were treated with 1 mM APMA for 30min before electrophoresis (lanes pG). As standards, reduced pre-stained Mr marker proteins (lane M), which electrophoresed withthe apparent Mrs indicated (in thousands), and human gingivalfibroblast 72-kDa gelatinase, both before (lanes F) and after (lanespF) APMA activation, were included.

alone or in assay buffer containing one of the followingreagents or combinations of reagents: 5 mM N-ethylmaleim-ide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10mM EDTA, 10 mM dithiothreitol (DTT), or 10 mM DTT plus10 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 adecrease in the molecular mass of the human gingivalfibroblast 72-kDa gelatinase from 66 kDa under nonreducingconditions (7, 35) to 62 kDa (Fig. 2). It is notable that theproenzyme form of gelatinase is activated during electropho-resis by the sodium dodecyl sulfate (4) present in thepolyacrylamide gels and therefore is also visualized as aclear band on the enzymograms with a molecular masscorresponding to the zymogen form of the enzyme. A134-kDa gelatinase present in the human gingival fibroblastconditioned medium was absent after APMA treatment andmost likely represents a dimer of the 72-kDa gelatinase. Wehave previously observed that APMA activation sometimesslightly reduces the net activity of collagenase obtained fromgingival crevicular fluid (9, 17), possibly through APMA-induced autolysis, and that APMA also induces the rapidautodegradation of rat 72-kDa gelatinase (33a). In this studytoo, APMA appeared to reduce the total amount of 92-kDagelatinase after activation. However, the thiol blocker NEMslightly 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-

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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 gelatinasewas purified to high specific activity by elution of the bound materialwith 4x concentrated electrophoresis sample buffer as describedin the text. Aliquots from the lysate and the gelatin-Sepharoseunbound material, the column wash, 1.0 M NaCl, and 4x electro-phoresis sample buffer containing 8% (wt/vol) sodium dodecylsulfate (SDS) and 8.0 M urea as eluants were analyzed by enzymog-raphy. Lane MGCF, monkey gingival crevicular fluid sample; laneMPDLF, gelatin-Sepharose affinity-purified gelatinase from monkeyperiodontal ligament fibroblasts. The apparent Mrs (in thousands) ofthe indicated gelatinases are shown. p-gelatinase, progelatinase.Serum albumin (A) and the electrophoresis front (f) are indicated.

molecular-mass gelatinases were totally blocked by thedivalent cation chelator EDTA and by purified human fibro-blast tissue inhibitor of metalloproteinases (TIMP; kindlyprovided by T. Cawston [Addenbrooke's Hospital, Cam-bridge, United Kingdom]) (data not shown) but not by theserine proteinase inhibitor PMSF. Thus, the decrease in Mrupon APMA treatment, the divalent cation requirement foractivity, the inhibition by TIMP, and the lack of inhibition byPMSF indicate that the 92-kDa gelatinase is a metalloprotei-nase and is therefore likely to be MMP-9, the 92-kDagelatinase in the MMP family of enzymes. The identities ofthe higher-M, gelatinases were not established, but they mayhave also been released from polymorphonuclear leukocytes(see below).

In an attempt to activate and reveal the presence of anybacterial thiol proteinases in the crevicular fluid, enzymo-gram incubations were performed either in the presence ofDTT alone or with DTT plus EDTA. However, there was noevidence for any gelatinolytic (Fig. 2) or caseinolytic (datanot shown) activities under these incubation conditions,indicating that these bacterial proteinases either were notpresent in the crevicular fluid or, unlike the 72- and 92-kDagelatinases, had become irreversibly inactivated either in thecrevicular fluid or during electrophoresis.

Since human gingival fibroblast gelatinase has an Mr of72,000 (under reducing conditions), it was unlikely thatunstimulated gingival or periodontal ligament fibroblastswere the source of the 92-kDa gelatinase. However, toensure that the 92-kDa gelatinase was not normally derivedfrom fibroblasts in the monkey, monkey periodontal liga-ment fibroblasts (24) were grown to confluence and madequiescent by serum deprivation for 24 h, and the conditionedmedium was collected after another 24 h. Gelatinase wasaffinity purified by gelatin-Sepharose chromatography asdescribed before (33, 35). Like human gingival fibroblasts,monkey periodontal ligament fibroblasts produced 72-kDagelatinase (Fig. 3, lane MPDLF), some of which was acti-vated during purification, as evidenced by the generation ofa lower-molecular-mass 64-kDa gelatinase.

Consistent with previous reports that polymorphonuclear

leukocytes are present in high numbers in periodontitis, thegingival tissues were found to be heavily infiltrated bypolymorphonuclear leukocytes after tooth ligation (27a).Polymorphonuclear leukocytes are known sources of pro-teolytic enzymes (13, 27, 28, 42, 47), and polymorphonuclearleukocyte-derived ,B-glucuronidase, lactate dehydrogenase,and arylsulfatase have been found in gingival crevicular fluid(19, 20). Therefore, to determine whether polymorphonu-clear leukocytes were a possible source of the 92-kDagelatinase, monkey polymorphonuclear leukocyte gelatinasewas affinity purified by gelatin-Sepharose chromatography.Polymorphonuclear leukocytes were prepared from heparin-ized whole-blood samples from each monkey by dextransedimentation (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 byultrasonication in distilled water, and the lysates were clar-ified by centrifugation (13,000 x g, 10 min) before beingloaded as either 1- or 2-ml samples onto 500-,ul minicolumnsof gelatin-Sepharose 4B (33, 35). The unbound material wascollected, and each column was washed with 2 ml of assaybuffer. Gelatin-Sepharose-bound material was then collectedby elution, first with 250 ,ul of 1.0 M NaCl in assay buffer andthen with 4x concentrated electrophoresis sample buffer(33, 35). As shown in Fig. 3, polymorphonuclear leukocytegelatinase present in the electrophoresis sample buffer eluantelectrophoresed with an apparent Mr of 92,000 and comi-grated with the major gelatinase present in the gingivalcrevicular fluid samples. In addition, minor polymorphonu-clear leukocyte gelatinases were present at 116 kDa and at ahigh, undetermined molecular mass, similar to the minorgelatinolytic species that were variably observed in thegingival crevicular fluid samples (Fig. 1). This indicated thatboth the 92-kDa and the high-molecular-mass gelatinasespresent in the gingival crevicular fluid were not of fibroblastor osteoblast origin and may have been derived from poly-morphonuclear leukocytes. In particular, the presence of the116-kDa gelatinase, a characteristic of polymorphonuclearleukocyte gelatinase activities, in the crevicular fluid sam-ples was demonstrated more often than not (Fig. 1), provid-ing further evidence for a polymorphonuclear leukocyteorigin for the 92-kDa gelatinase. Moreover, the occasionalpresence of small amounts of the 72-kDa gelatinase (electro-phoresing at 66 kDa under nonreducing conditions) furtherindicates the minor contribution of fibroblastic MMPs in thisexudate.Although comigration of proteins by electrophoresis does

not confer identity, additional evidence for a polymorpho-nuclear leukocyte origin for the collagenolytic enzymespresent in gingival crevicular fluid was derived from exper-iments using antibodies known to block the activity offibroblast collagenase but not that of leukocyte collagenase(3). Procollagenase in the gingival crevicular fluid sampleswas activated with 1.0 mM APMA for 30 min at 22°C (32)before the addition of either rabbit anti-human fibroblastcollagenase antibody at various concentrations from a 1/2 toa 1/200 dilution (antiserum was kindly provided by H.Birkedal-Hansen, Department of Oral Biology, University ofAlabama, Birmingham) or nonimmune serum antibody, bothpurified on protein A-Sepharose. After incubation at 22°C for45 min, the collagenase assay was initiated by the addition of2,500 dpm (-7 ng) of metabolically labeled [14C]glycine typeI collagen (32). After an 18-h incubation at 22°C, the assayreaction products were resolved by electrophoresis on 7.5%polyacrylamide gels (32). For positive controls, human gin-

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FIG. 4. Effect of neutralizing anti-human fibroblast collagenaseantibody on gingival crevicular fluid collagenase activity. Monkeygingival 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 describedin the text. After the assay, the reaction products were resolved byelectrophoresis on 7.5% polyacrylamide gels, and the [14C]glycine-labeled al- and a2-collagen chains and the collagenase-generated3/4-collagen (tA) bands were visualized by fluorography after expo-

sure to Kodak SB5 film at -70°C. For controls, purified humangingival 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 forcollagenase inhibition. Lane S, type I collagen substrate controlincubated in the absence of collagenase-containing samples.

gival fibroblast and monkey periodontal ligament fibroblastcollagenases were partially purified from 24-h conditionedcell 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) wasalso used as a control. Purified human fibroblast TIMP wasused as a positive control for collagenase inhibition. Theoccasional 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 notspecifically 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 collagenaseantibody totally inhibited human gingival fibroblast collage-nase activity, it had no effect on the collagenase activitypresent in the crevicular fluid samples at concentrations ashigh as a 1/2 dilution of the original volume of antiserum(Fig. 4). That this was not due to species differences inantibody reactivity was shown by the inhibition of monkeyperiodontal ligament fibroblast collagenase activity. Thus,together with the absence of detectable bacterial collagena-ses, the lack of inhibition of monkey gingival crevicular fluidcollagenase activity by the anti-human fibroblast collagenaseantibody provides further evidence that polymorphonuclearleukocytes 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 polymorphonuclearleukocyte source for gingival crevicular fluid collagenase hasaccumulated. However, substrate specificity and enzymeactivation 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 crevicularfluid samples further complicates the interpretation of thesedata, since the collagenase inhibitors complex with theactivated MMPs and only the net levels of activated colla-

genase are then detectable (35). In addition, depending onthe method of activation, different degrees of autodegrada-tion of the activated MMPs may be observed, adding furthervariability to the data. Thus, while enzyme activation andsubstrate specificity analyses are useful, they only provideindirect evidence for collagenase typing. Until blockingantibodies to leukocyte collagenase become generally avail-able, the use of antibodies to fibroblast collagenase alsoprovides only indirect, though compelling, evidence for apolymorphonuclear leukocyte origin for crevicular fluid col-lagenase.Other than the minor amounts of 72-kDa gelatinase that

were occasionally present in the gingival crevicular fluidsamples, connective tissue cell-derived MMPs could not befound 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 basisof activation characteristics, latent interstitial collagenasewas found to predominate over leukocyte collagenase atsites of treated periodontitis (9). It is conceivable thatactivated collagenase and other MMPs at the focus ofinflammation are eliminated by autodegradation (3), by re-maining bound to insoluble collagen (34, 45), by complexingwith inhibitors, or by being cleared through the lymphaticsrather than by being cleared into the crevicular fluid.

Overall, the data presented here strongly implicate poly-morphonuclear leukocytes as the major cellular source ofcollagenolytic proteinases in the gingival crevicular fluidduring episodes of inflammation associated with connectivetissue destruction. Moreover, until now, the presence, iden-tity, and source of gelatinases in gingival crevicular fluid wasunknown. The function of polymorphonuclear leukocytes inthe 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). Polymorphonuclearleukocytes leave the gingival vascular plexus and migratethrough the tissue to the gingival sulcus, where they preventmicrobial invasion of the periodontium by phagocytosis andby the release of lysosomal enzymes. However, since poly-morphonuclear leukocyte degranulation occurs both in theperiodontal 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 thedegradative process. Indeed, our previous work has re-vealed a positive association between the presence of activecollagenase and periodontal attachment loss (22) and anincreased severity of disease (9, 17, 32, 34). Moreover, afterthe resolution of periodontal inflammation, the levels ofactive collagenase in the gingival crevicular fluid decrease (9,11, 21, 22). Thus, analysis of the proteinases that may beresponsible for the destruction of the major structural pro-teins in tissues provides a rational approach toward under-standing the underlying mechanisms of connective tissuedestruction during inflammation.

C.M.O. is supported by a Medical Research Council of CanadaCentennial Fellowship. C.A.G.M. is supported by a Career ScientistAward from the Ontario Ministry of Health. This work was sup-ported by grants from the Medical Research Council of Canada andthe Ontario Ministry of Health.

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