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474

Role of Matrix Metalloproteinasesin Human Periodontal Diseases*Henning Birkedal-Hansen

Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that me-diate the degradation of extracellular matrix macromolecules, including interstitial andbasement membrane collagens, fibronectin, laminin, and proteoglycan core protein. Theenzymes are secreted or released in latent form and become activated in the pericellularenvironment by disruption of a Zn++-cysteine bond which blocks the reactivity of theactive site. The major cell types in inflamed and healthy periodontal tissues (fibroblasts,keratinocytes, endothelial cells, and macrophages) are capable of responding to growthfactors and cytokines, as well as to products released from the microbial flora by inductionof transcription of 1 or more MMP genes. Cytokines that are likely to regulate expressionof MMP

genes

in

periodontaltissues include IL-1, TNF-a, and TGF-a. In addition,

triggered PMN leukocytes which express only 2 MMP (PMN-CL and Mr 92K GL) releasethese enzymes from specific granule storage sites in response to a number of stimuli.The evidence that MMP are involved in tissue destruction in human periodontal diseasesis still indirect and circumstantial. Cells isolated from normal and inflamed gingiva arecapable of expressing a wide complement of MMP in culture and several MMP can bedetected in cells of human gingiva in vivo. In addition, PMN-CL and Mr 92K GL arereadily detected in gingival crevicular fluid from gingivitis and Periodontitis patients.Osteoclastic bone resorption does not appear to directly involve MMP, but a body ofevidence suggests that bone resorption is initiated by removal of the osteoid layer byosteoblasts by means of a collagenase-dependent process. J Periodontol 1993; 64:474-484.

Key Words: Enzymes, proteolytic; cytokines; growth factors; periodontal diseases/path-ogenesis; bone resorption; leukocytes; proteinases.

METABOLIC DEGRADATION OF THEEXTRACELLULAR MATRIX

Degradation of the extracellular matrix may involve as manyas 4 apparently distinct pathways (Fig. 1). A body of evi-dence suggests that matrix components may be dissolvedby extracellular matrix metalloproteinase (MMP)-depen-

dentor

plasmin (Pln)-dependent cleavage reactions, andthat larger fragments of matrix may be disposed of by aphagocytic pathway by way of cleavage by lysosomal pro-teinases. Mineralized matrices appear to be degraded by acomplex extra/pericellular process mediated by osteoclasts(osteoclastic pathway) which relies on degradation by ly-sosomal proteinases in a narrow pericellular compartment.This review focuses specifically on matrix metalloprotein-ases and their role in the metabolic degradation of the ex-tracellular matrix in human periodontal diseases. A com-prehensive review of the matrix metalloproteinase fieldincluding a complete bibliography of the literature through

*Department of Oral Biology and Research Center in Oral Biology, Schoolof Dentistry, University of Alabama at Birmingham, Birmingham, AL.

Figure 1. Pathways for degradation of the extracellular matrix. Areas ofoverlap are indicated by boxes.

1990 compiled by Woessner1 was recently published.2 Forshorter general discussions of the role of MMP in healthand disease the reader is referred to recent reviews.3,4 Atthe outset it is important to recognize that much of theevidence for the role of MMP in specific developmental orpathological processes remains circumstantial and indirect.That is certainly true for the involvement of MMP in human

periodontal diseases. It has proven difficult to specificallyidentify the natural substrates for most MMP in part becauseof their relatively broad and overlapping substrate specific-

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Volume 64Number 5 BIRKEDAL-HANSEN 475

ities (Table 1). Moreover, the relationship of the MMP-dependent pathway to other routes of matrix degradationhas not been adequately clarified. Yet the observation thatMMP transcripts or protein frequently are present in cells,tissues, and interstitial fluids in vivo strengthens the notionthat these enzymes are involved in the metabolic remod-

eling of the extracellular matrix.

Matrix MetalloproteinasesThe MMP gene family encodes 9 or more metal-dependentendopeptidases which collectively are capable of degradingmost, if not all, extracellular matrix macromolecules (Table1). The enzymes share extensive sequence homology butdiffer somewhat in terms of substrate specificity and tran-scriptional regulation. All MMP may be regarded as deriv-atives of a 5-domain prototype structure, formed either byaddition or deletion of regulatory domains as shown in Fig-ure 2. A short

signal sequenceis followed in order

bya

propeptide which endows the virgin enzyme with catalyticlatency, a catalytic domain which contains the active siteand the catalytic machinery, a proline-rich hinge region,and a pexin-like COOH-terminal domain which plays a rolein determining substrate specificity. In addition, the 2 ge-latinases contain a gelatin-binding insert (fibronectin typeII-like repeats) in the catalytic domain.5*6 Each of the en-zymes contains a tridentate Zn++-binding site believed toconstitute the active site.7 The pexin-like domain is absentfrom the smallest MMP, PUMP-1.

The PMN-CL gene is only expressed by PMN leukocytes

whereas the highly homologous FIB-CL gene is expressedby fibroblasts,8*9 keratinocytes,10,11 endothelial cells,12*13

monocytes/macrophages,14*15 osteoblasts,16*17 and chondro-cytes.18 The highly glycosylated PMN enzyme has a con-siderably larger molecular mass than FIB-CL (Mr 75,000vs. 57,000/52,000) but the 2 protein cores are of virtuallyidentical size. The 2 collagenases differ markedly in termsof transcriptional regulation. PMN-type collagenase is rap-idly released from specific granule storage sites of triggeredPMNs whereas FIB-CL is synthesized on demand by ini-tiating transcription of the corresponding gene. This processcauses a lag period of 6 to 12 hours before enzyme can bedetected in the extracellular environment.

The stromelysin group of MMP includes SL-1 and SL-2which have virtually identical substrate specificities and cleavea wide range of extracellular matrix proteins (proteoglycancore protein, type IV and V collagen, fibronectin, and lam-inin.)19 SL-1 is expressed by stromal cells either constitu-tively or after induction by growth factors/cytokines [IL-

l,20 EGF,21 TNF-a,20 PDGF21]or

phorbolesters.22*23 The

enzyme does not appear to be expressed by PMN leukocytesand keratinocytes in the human, but a SL-1 (or SL-2) homo-logue is induced in murine skin epidermis by treatment withthe tumor promoter TPA (12-O-tetradecanoyl-phorbol ace-tate).23*24 SL-2 is expressed less abundantly than SL-1 anddoes not appear to respond to growth factors (EGF and IL-1) or phorbol esters.

The Mr 72K GL is perhaps the most widely distributedof all MMP and has been identified in fibroblasts,25 kera-tinocytes,26 endothelial cells,27 monocytes/macrophages,28osteoblasts,29 and chondrocytes.30 Mr 72K GL does not

appear, however, to be expressed by PMN leukocytes butis present in a circulating form in plasma.31 The Mr 92K

Table 1: Matrix Metalloproteinase Family

Enzyme Abbreviation MMP# MrExtracellular Matrix

Substrates

Interstitial CollagenasesFibroblast-type FIB-CL

CollagenasePMN-type PMN-CL

Collagenase

StromelysinsStromelysin-1 SL-1

Stomelysin-2 SL-2

GelatinasesMr 72K Gelatinase/ Mr 72K GL

Type IV CollagenaseMr 92K Gelatinase/ Mr 92K GL

Type IV Collagenase

Other

Stromelysin-3 SL-3PUMP-1 PUMP-1

Macrophage Metallo- MMEelastase

MMP-1

MMP-8

57,000/52,000

75,000

MMP-3 60,000/55,000

MMP-10 60,000/55,000

MMP-2 72,000

MMP-9 92,000

MMP-11 n.d.MMP-7 28,000

55,000

Collagen I, II, III, VII, VIII, X,Gelatin

Same as FIB-CL

PG Core Protein, Fibronectin,Laminin, Collagen IV, V,IX, X, Elastin

Same as SL-1

Gelatin, Collagen IV, V, VII, X,Elastin, FibronectinGelatin, Collagen IV, V, Elastin

n.d.Fibronectin, Laminin,

Collagen IV, Gelatin, proCL,PG Core Protein

Elastin

MMP numbering according to Nagase et al.13

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476 MATRIX METALLOPROTEINASESJ Periodontol

May 1993 (Supplement)

PROTOTYPE

Mr 72 K GL,Mr 92 K GL

SL-1,-2,-3PMN-CL, FIB-CL

HINGE

PROPEPTIDE CATALYTIC DOMAIN,

PEXIN-LIKE DOMAIN

FIBRONECTIN-LIKE PROLINE-HICHINSERT

HINGE

Table 2. Transcriptional Effect of Growth Factors and Cytokines onMatrix Metalloproteinases

Induction

IL-1 a,TNF-a

TGF-aEGFPDGFbFGFNGFTGF-

Reference

20, 4520, 46, 47

4921

21, 4844, 5051

26, 41

Repression

TGF-IFN-7

Reference

50, 5253, 54

PUMP-1

Figure 2. Domain structure of matrix metalloproteinases.

GL is produced by PMN leukocytes,32'33 keratinocytes,26'34and

monocytes/macrophages,35and

occasionally by fibro-blasts. It is interesting to note that PMN which express aunique and distinct CL gene utilize the same Mr 92K GLgene as other cells although in a manner which yields astorable rather than a secreted enzyme. Mr 72K GL is ex-

pressed constitutively by most cells in culture and respondsonly moderately to growth factors which induce the Mr 92KGL. The Mr 72K and Mr 92K GL cleave a number of

peptide bonds in denatured collagen chains to yield smallpeptides.25'36 Besides gelatin, gelatinases also cleave typesIV, V, VII, and X collagens37"39 and elastin.40

PUMP-1 cleaves a wide range of substrates including

fibronectin, laminin,and

gelatin.The

enzymeis

expressedin gingival fibroblasts41 and has been isolated from the in-voluting rat uterus and from rectal carcinoma cells.42,43 SL-3transcripts are expressed by mesenchymal cells of humanmammary carcinomas, often adjacent to invading epithelialtumor cells. SL-3 expression is also induced in embryonicfibroblasts by several growth factors. It is unknown whetherSL-3 is produced by cells of human periodontal tissues.The amino acid sequence deduced from cDNA suggests thatSL-3 encodes a functional metalloproteinase, but the en-zyme protein has not yet been isolated or characterized.SL-3 has the same principal domain structure as CLs andSL-1 and SL-2, but differs

byan insert of 10 residues at

the autolytic activation site.44

Regulation of MMP ActivityThe activity of MMP against extracellular matrix substratesis regulated at 4 "gates": 1) by transcriptional regulationof MMP genes; 2) by precursor activation; 3) by differencesin substrate specificity; and 4) by MMP inhibitors.

Transcriptional regulation. The transcription of mostMMP genes is regulated by endogenous growth factors andcytokines (Table 2). Stimulation or repression of growthfactor- and cytokine-responsive MMP genes results in up

toa

50-fold change in mRNA and protein levels. Tran-scription of the CL and SL-1 genes (and in some cells theSL-3 and Mr 92K GL genes as well) is induced by IL-

Propeptido

LATENT ACTIVE

Figure 3. Activation of MMP precursors is initiated by opening of thecysteine switch.

I,20,45 TNF-a,20'46'47 PDGF,21,48 TGF-a,49 EGF,21bFGF,20,50 NGF,51 and with few exceptions26 abrogated byTGF-50,52 and IFN-7.53,54 TGF- which ablates transcrip-tion of CL and SL-150,52 upregulates Mr 72K GL by 2- to4-fold and Mr 92K by up to 8-fold.26,41 These findings areof considerable interest because TGF- in general appearsto down-regulate rather than stimulate MMP expression.

Transcription of MMP genes may also be regulated by otherendogenous pathways. FIB-CL is induced or stimulated inosteoblasts by PTH and 1,25 di(OH)D3,17,55'56 and FIB-CLand SL-1 are down-regulated by glucocorticoids and byretinoids.57 59 The proximity of a viable microbial plaque,moreover, creates an unequaled opportunity for direct tran-scriptional effects by microbial metabolites on human gin-gival and periodontal cells. A potentially important inter-action that falls in this category is the induction of FIB-CLin macrophages by LPS.60'61 In addition, microbialproteinases62,63 and lectins64 may also play a role in thetranscriptional regulation of MMP expression.

Activation ofprecursors. The latency of MMP precur-sors appears to be maintained, at least in part, by a coor-dinate bond which links an unpaired Cys residue in thepropeptide to the active site Zn++65,66 (Fig. 3). Disruptionof the Cys-Zn++ bond is a prerequisite to activation andmay be achieved in a number of different ways: 1) byinteraction or modification of the Cys residue by organ-omercurials, metal ions, thiol reagents, and oxidants; 2) byconformational change of the Polypeptide backbone in-duced by certain chaotropic agents (KI, NaSCN) and de-tergents (SDS); and 3) by excision of a portion of the pro-pepeptide by proteolytic enzymes (trypsin, plasmin,

chymotrypsin, neutrophil elastate, cathepsin B, and plasmakallikrein). The enzyme subsequently catalyzes 1 or moreautolytic cleavages to generate the fully-processed form.66 69

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Although MMP can readily be activated in the test tube byany or all of these mechanisms, the biologic activationmechanisms are still poorly understood. Several studies havesuggested that plasmin may play a role in activation of FIB-CL and SL-1, at least in cell culture systems, and that SL-

1 is perhaps involved in the processing of the FIB-CL pre-cursor to a particularly highly-active form.70-72 There is stillinsufficient evidence to determine whether these reactionsalso take place in vivo. It is of note, however, that thedegradation of gelatin and collagen by PMN leukocytes73'74involves activation of CL and Mr 92K GL by oxidativepathways, presumably by oxidation of the unpaired pro-peptide Cys residue by HOC1.

Substrate Specificity. A certain level of regulation ofMMP activity is encoded at the level of the substrate. Al-though the enzymes have somewhat overlapping substratespecificities, there are also notable differences, particularlywith respect to the cleavage of collagens.2 Virtually all ofthe enzymes cleave gelatin and fibronectin at some rate,and most cleave type IV and V collagens at sufficientlyhigh temperatures. The characteristic ability of FIB-CL andPMN-type collagenases to dissolve interstitial collagen fi-brils by cleavage of the component molecules in the pro-teinase-resistant triple-helical domain, however, is not sharedby other members of the family.

Inhibition

a-Macroglobulins. Active MMP are captured by a-ma-croglobulins by a unique venus-fly-trap mechanism acti-vated by cleavage of a bond in the "bait region."75,76 Thiscleavage leads to hydrolysis of a labile internal thiol-esterbond and covalent cross-linking of a nascent glytamyl res-idue to lysyl side chains exposed on the surface of theattacking proteinase.77 The rapid capture rates, particularlywith CL, suggest that a-macroglobulins, particularly al-M, play an important role in the regulation of MMP activity.

Tissue inhibitors of metalloproteinases (TIMPs). TIMPSform classical non-covalent bimolecular complexes with theactive forms of MMPs and, in some instances, with latentMMP precursors as well. TIMPs appear to regulate matrix

degradationboth

by proteinaseelimination and

by blockageof autolytic MMP activation.78,79 Two members of the TIMPfamily, TIMP-1 and TIMP-2, have been identified but moremay exist.13,80"83 TIMPs are widely distributed in tissuesand fluids84,85 and are expressed by many cell types in-cluding fibroblasts,86 keratinocytes,87 monocytes/macro-phages,15 and endothelial cells.13 TIMP-1 is a Mr 28K gly-cosylated protein with a Mr 20K protein core.80,86 TIMP-1forms complexes with active collagenase, but not procol-lagenase, in a 1:1 stoichiometry with rCjS of 10~9M to10" 10M.88,89 Complex formation with active MMP does notappear to depend on cleavage of the inhibitor and fully

functional inhibitor can be recovered from the complex.90TIMP-1 also forms a complex with the zymogen form ofMr 92K GL.5,79,91 TIMP-2, a Mr 22,000 unglycosylatedprotein, is expressed by fibroblasts and endothelial cells and

perhaps by other cells as well.13,92,93 The inhibitor forms acomplex with the zymogen form of Mr 72K GL.5,89 TIMP-2 is 2- to 10-fold more effective than TIMP-1 against the2 GL whereas TIMP-1 appears to more effectively inhibitCL.91 The 2 TIMP genes are differently regulated. TIMP-1

expression is stimulated by growth factors (EGF,TNF-

a, IL-1, TGF-), phorbol esters, retinoids, and glucocor-ticoids, whereas TIMP-2 expression is down-regulated byTGF- and fails to respond to phorbol esters.41,94 98

ROLE OF MMP IN HUMAN PERIODONTAL

DISEASES

Expression of MMP by Different Cell Types of HumanPeriodontal Tissues

Each of the major cell types of human periodontal tissuesis capable of expressing a unique complement of MMP

when properly stimulated as summarized in Table 3. PMNleukocytes share with many other cell types the ability toexpress the common 92K GL gene but, for reasons that areunknown, utilize a distinct gene for interstitial collagenase(PMN-CL). Both enzymes are stored in specific granulesand rapidly released when the cells are triggered.99 Fibro-blasts express a somewhat larger complement of MMP,including an interstitial, fibroblast-type collagenase (FIB-CL), SL-1, SL-3, Mr 72K GL, and PUMP-1, but not Mr92K GL. Keratinocytes express FIB-CL, Mr 72K GL, andMr 92 K GL, but not SL-1. Macrophages and endothelialcells each express FIB-CL, SL-1, and both the Mr 72K and

the Mr 92K GL.12,13,20,39

Cell Type-Specific Differences in Regulation of MMPThe synthesis and packaging of MMP in specific granulesare essentially complete when the PMN enters the bloodstream. The triggered release of MMP from specific gran-ules is clearly not a transcriptional event and it is still un-certain to what extent it is regulated by growth factors andcytokines. The response pattern of the PMN leukocyte isclearly unique and different from that of any other cell typenot only in the complement of MMP expressed but also inthe utilization of the

enzymes.The PMN

leukocytehas

evolved to respond rapidly and in full force to externalstimuli. No other cell type is capable of "dumping" suchlarge quantities of destructive enzymes in a matter of min-utes. The response is sustained, when needed, by contin-uous recruitment of new cells and not by prolonged releasefrom cells already triggered. Fibroblasts, keratinocytes,macrophages, and endothelial cells share an essentiallycommon response to catabolic growth factors such as IL-1, TNF-a, TGF-a, EGF, bFGF, PDGF, namely inductionor stimulation of transcription of MMP genes. However,there are important cell-specific and cytokine-specific dif-

ferences among these responses. In fibroblasts, IL-l in-duces primarily SL-1, TNF-a primarily CL,20 IFN-7 re-presses SL-1 but moderately upregulates CL,53 TGF-represses CL and SL-1, but upregulates 72K GL. In kera-

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Table 3. Expression of Matrix Metalloproteinases by Five Major Cell Types of Human PeriodontalTissues

Cell TypePMN-

Leukocyte Fibroblast Keratinocyte MacrophageEndothelial

Cell

Enzymesexpressed

Transcriptionalactivation

Mobilizationof enzymes

Responsetime

Responseduration

PMN-CL,Mr 92K GL

GranuleRelease

Seconds

Minutes

FIB-CL, SL-1,Mr 7K GL,

PUMP-1,SL-3

IL-1, TNF-a,EGF, TGF-a,PDGF, TPA


6 to 12 hours 6 to 12 hours 6 to 12 hours

Days

FIB-CL,Mr 72 K GL,

Mr 92K GL,SL-2

TNF-a, EGF,TGF-a,TGF-,TPA



Mr 92K GL

TPA



Mr 92K GL

TPA


6 to 12 hours

Days

tinocytes, TGF- upregulates both the Mr 72 K and the Mr92K GL;26 on the other hand, IL-l, which is the mostpotent known inducer of CL- and SL-l-expression by fi-broblasts, has no effect on keratinocytes, at least not in thehuman.11 Recent studies in our own laboratory have shownthat growth factors and cytokines which induce expressionof SL-1 in stromal cells induce exclusively SL-2 in kera-tinocytes (unpublished data). The findings summarized aboveshow: 1) that different cell types express different comple-ments of MMP; 2) different cytokines elicit different tran-scriptional effects in the same cell type; and 3) that different

cell types do not necessarily respond in the same fashionto a given cytokine. In the light of these findings, and inthe light of the many potential synergistic and antagonisticinteractions that exist between 2 or more cytokines, it isapparent that different inflammatory infiltrates may varyconsiderably in destructive potential. This is perhaps thekey to understanding why inflammation of the human gin-giva may or may not give rise to tissue destruction andattachment loss.

Recent studies have started to shed light into the mech-anisms that enable growth factors and cytokines to elicitoverlapping, yet occasionally distinct, transcriptional ef-fects. The AP-1 binding site is a necessary, but not suffi-cient, requirement for transcriptional activation of MMPexpression by many growth factors and cytokines. For in-stance, the Mr 92K GL gene which contains 3 copies ofthe AP-1 site is inducible by catabolic growth factors whereasthe Mr 72K GL gene which contains no such elements isnot.100-101 The transcriptional activity of the AP-1 site de-pends on binding of c-fos/c-jun dimers to the TGAGTCAsequence. The role of fos, however, is quite complex andeven among transcriptional pathways that operate throughthe AP-1 site, some but not all, are c-fos-dependent. Forinstance, induction of SL-1 by PDGF is fos-dependentwhereas that of EGF is not.21 Moreover, abrogation of FIB-CL and SL-1 transcription by TGF- is also dependent onc-fos although the action of this growth factor is indepen-

dent of the AP-1 site and instead depends on a unique 10bp TGF--responsive element.50,52 Recent studies haveidentified promoter and enhancer elements required formaximal transcriptional activation that are specific for eachMMP. Such is the case for the PEA-3 site in the FIB-CL

promoter and the NIP protein binding site in the SL-1 pro-moter.102"104

The second messenger-signaling pathways which me-diate growth factor and cytokine transcriptional effects onMMP gene expression are still incompletely understood. Abody of evidence suggests that protein kinase C (PKC), a

cellular receptor for TPA, acts as a messenger in the tran-scriptional regulation of growth factor-responsive MMP genesin some but not all cases.53,105 cAMP may be involved asa second messenger in some responses but apparently throughactivation of a different (Jun B-dependent) transcriptionalmachinery than either TPA or growth factors/cyto-kines.58,106 cAMP stimulates MMP expression in some celltypes; in others leads to repression. For instance, expressionof CL and SL-1 by guinea pig peritoneal macrophages, ratUMR-106 osteosarcoma cells, and rat fibroblasts is stim-ulated by cAMP,24,56,107 whereas the constitutive expres-sion of CL by human GM637 fibroblasts,104 and the IL-1,EGF- and oncogene-induced transcription of FIB-CL andSL-1 genes by rat and human fibroblasts is repressed.52,108

What Are the Important Cytokines and GrowthFactors?The complexity of the cytokine networks and the manydifferent cellular responses among resident and immigrantcells of inflamed periodontal tissues give rise to an almostunlimited number of putative pathogenic processes. Clearlynot all of these are equally important, but it is not yetpossible to identify those cellular interactions that actuallydrive attachment loss. It is likely that IL-l plays an im-portant role in regulating MMP expression in inflamed hu-man gingiva. IL-la and - are present in concentrations of10"9-10_8M109,110 which are clearly sufficient to induce

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Table 4. Tissue Destructive Enzymes and Inhibitors in Inflammatory Exudates, Saliva, and Plasma

Enzyme/Inhibitor FluidConcentration

M-g/ml Reference

Collagenase(PMN)

Mr 92K gelatinaseMr 72K gelatinase

Plasminogen

TIMP

a2-Macroglobulin

GCF

Saliva

PlasmaGCF

GCF

Plasma

Synovial fluidPlasmaGCF

Synovial fluidGCFPlasma

1-10

0.340

0.5-0.60.1-0.5

40-200

2000.3-4.61.0-1.50.2-1.0

700-1100

200-2,4002,250

115, Estimate

127

31Estimate

Estimate

140

141, 142128Estimate

143

120, 121, 122143

expression of SL-1 and CL in cultured fibroblasts. (10~9-1010 M). IL-1 is also a potent inducer of bone resorption,but it is not known whether this response is related to theinductive effect of IL-1 on MMP. TNF-a is also present in

inducing concentrations in the range 0.1-13 nM.111 It isreasonable to assume that TGF-a may also be involved,although the concentration of TGF-a in GCF has not yetbeen determined. TGF-a is a potent inducer of CL and Mr92K GL in keratinocytes, and recent studies have shownthat the EGF-receptor (which also serves as a receptor forTGF-a) is expressed abundantly on epithelial cells of theoral mucosa.112

MMP Are Expressed by Gingival/Periodontal Cells InVivo

Woolley and Davis113 investigated the expression of MMPin normal or inflamed human gingiva by immunolocaliza-tion methods. FIB-CL was associated with cells in the gin-gival connective tissue often in close proximity of the junc-tional and pocket epithelia but not in any epithelial cells.Enzyme was also associated with collagen fibrils and oc-casionally with inflammatory infiltrates. Our own studieshave shown that only low levels of MMP are expressed inany one section. Cells scattered throughout the connectivetissue (fibroblasts and/or macrophages) show staining of the

perinuclear region with antibodies to FIB-CL. Mr 72K GLis not infrequently associated with blood vessels and cap-illaries. Even heavily-inflamed human gingiva shows onlysporadic expression of MMP with no discernible pattern ofdistribution (unpublished data). In the aggregate, immu-nolocalization studies have proven fairly inconclusive andhave given few, if any clues, to the level of involvementof MMP in periodontal diseases.

MMP in GCF and Saliva

GCF and saliva contain at least 2 MMP, CL, and Mr 92KGL.114"116 CL appears to be exclusively or predominantlyof the PMN-type.117,118 The apparent absence of SL-1 andMr 72K GL under conditions that readily permit detection

of Mr 92K GL leads us to believe that the Mr 92 K GL,too, is of PMN origin. These observations suggest that PMNleukocytes are the dominant, and perhaps the only signifi-cant, contributors of MMP in GCF. If so, the questionarises whether the enzymes are indeed released from PMNsin the tissue or the crevicular fluid, or perhaps still insidethe cells at the time of sampling and only secondarily re-leased as a sampling artifact. "GCF enzymes" producedby PMN leukocytes and perhaps carried to the GCF stillinside the cells include CL, Mr 92K GL, elastase, and

myeloperoxidase. Measurement of these activities in oralfluid samples appears to be essentially an enzyme-basedPMN count, a concern previously voiced by Cimasoni.119

Metabolites in GCF may originate from 3 different com-partments: from plasma, from resident cells/stroma, andfrom PMN leukocytes (Table 4). Serum components in-cluding a-2M, albumin, and Igs typically are present athigh concentrations, varying between 20% and 100% ofplasma values. a2-M concentrations, for instance, vary be-tween 0.2-2.4 mg/ml or 10 to 1000 times higher than anyof the MMP.120123 Because of the high concentration ofa2-M, it is unlikely that any MMP exists in GCF in anactive form. This notion apparently conflicts with the find-ing of more active (versus latent) MMP in Periodontitispatients/sites,123 126 but as previously alluded to it is un-certain whether "active" MMP in GCF exist as such or are

generated by solubilization or lysis of PMNs.Plasminogen is by far the most abundant proteinase in

GCF. Although the concentration of Plasminogen has notbeen accurately determined, we predict (based on the con-centration of other plasma proteins) that it is as high as 40to 200 |xg/ml. By comparison, we have previously esti-mated that the concentration of CL may reach 10 |xg/ml,but it is probably much lower. Since Mr 72K GL is presentin plasma at a concentration of approximately 0.5 (xg/ml,31we also predict that this enzyme, although undetectable byzymographic analysis, exists in GCF at a concentration of0.1 to 0.5 |xg/ml even in the absence of any local contri-bution from the tissues. Mr 92K GL, presumably of PMN

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origin, is present in saliva in relatively high levels (340 ng/ml).127 It is also readily detectable in GCF112 but the con-centration is not known. TIMP-1 (or TIMP-2) levels in GCFare unknown but based on the plasma concentration (1.0-1.5 u,g/ml)128 we estimate that TIMP concentrations of GCFare at least 0.2 to 1.0

Lig/ml.Metabolites that originate from the resident cell/stromapresumably are washed away by the outward fluid flowprovided they escape local capture and elimination. It islikely the PGE2, IL-1, and TNF-a are true metabolites re-leased from cells indigenous to the area or perhaps newly-recruited macrophages. Recent studies have shown, how-ever, that triggered PMN leukocytes activate transcriptionof the IL-1 gene.129 It therefore cannot be excluded thatPMN leukocytes are a major source of IL-1 in GCF.

Association of GCF MMP With Disease SeverityA number of studies have aimed at correlating MMP activ-ities with disease severity. Several experiments have shownthat when group means are compared, CL activity increaseswith disease severity (Periodontitis > gingivitis > health)and that treatment tends to return GCF values to controllevels.115'124-126 It has not been possible to convincinglydemonstrate similar correlations for individual sites, pre-sumably because of the high site-to-site, person-to-person,and day-to-day variation. Neither the "level" nor the "pat-tern" of activity at the site seems to correlate well with"bone loss" over a 6-month period.130,131 When monitor-ing a

particularsite over an extended

periodof time, the

high degree of week-to-week variation suggests that a singlemeasurement taken at a particular point in time has little,if any, predictive value. Sporadic "high" values never re-main high for long and do not necessarily herald impendingbone loss.130,131

Role of MMP in Bone ResorptionA number of studies have suggested that osteoblasts expressFIB-CL when stimulated by bone-resorbing agents.16,56 Theseobservations have led to the hypothesis that osteoclasticbone resorption is initiated by an Osteoblast response to

resorptive signals such as PTH, which includes expressionof FIB-CL and perhaps other MMP, and result in dissolu-tion of the unmineralized collagenous osteoid layer.132Osteoblasts later vacate the surface as newly-recruited os-teoclasts move in. Osteoclasts do not appear to expressMMP, but utilize a distinct acidic cathepsin-dependentmechanism for dissolution of mineralized matrices.133'134 Itis possible, although not yet proven, that expression ofMMP is an early key event in bone resorption, a findingthat may help explain why bone resorption is so highlysensitive to IL-1 and TNF-a.135138

AcknowledgmentsThis study was supported by NIH grants DE08228 andDE06028.

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Send reprint requests to: Dr. Henning Birkedal-Hansen, Department ofOral Biology, University of Alabama School of Dentistry, University ofAlabama at Birmingham, UAB Station, Birmingham, AL 35294.

birkedal 1993

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