474Role of Matrix Metalloproteinases in Human Periodontal Diseases* Henning Birkedal-Hansen Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix macromolecules, including interstitial and basement membrane collagens, fibronectin, laminin, and proteoglycan core protein. The enzymes are secreted or released in latent form and become activated in the pericellular environment by disruption of a Zn++-cysteine bond which blocks the reactivity of the active site. The major cell types in inflamed and healthy periodontal tissues (fibroblasts, keratinocytes, endothelial cells, and macrophages) are capable of responding to growth factors and cytokines, as well as to products released from the microbial flora by induction of transcription of 1 or more MMP genes. Cytokines that are likely to regulate expression of MMP genes in periodontal tissues include IL-1, TNF-a, and TGF-a. In addition, triggered PMN leukocytes which express only 2 MMP (PMN-CL and Mr 92K GL) release these 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 diseases is still indirect and circumstantial. Cells isolated from normal and inflamed gingiva are capable of expressing a wide complement of MMP in culture and several MMP can be detected in cells of human gingiva in vivo. In addition, PMN-CL and Mr 92K GL are readily detected in gingival crevicular fluid from gingivitis and Periodontitis patients. Osteoclastic bone resorption does not appear to directly involve MMP, but a body of evidence suggests that bone resorption is initiated by removal of the osteoid layer by osteoblasts by means of a collagenase-dependent process. J Periodontol 1993; 64:474484. Key Words: Enzymes, proteolytic; cytokines; growth factors; periodontal diseases/path- ogenesis; bone resorption; leukocytes; proteinases. METABOLIC DEGRADATION OF THE EXTRACELLULAR MATRIX Degradation of the extracellular matrix may involve as many as 4 apparently distinct pathways (Fig. 1). A body of evidence suggests that matrix components may be dissolved by extracellular matrix metalloproteinase (MMP)-dependent or plasmin (Pln)-dependent cleavage reactions, and that larger fragments of matrix may be disposed of by a phagocytic pathway by way of cleavage by lysosomal proteinases. Mineralized matrices appear to be degraded by a complex extra/pericellular process mediated by osteoclasts (osteoclastic pathway) which relies on degradation by lysosomal proteinases in a narrow pericellular compartment. This review focuses specifically on matrix metalloproteinases and their role in the metabolic degradation of the extracellular matrix in human periodontal diseases. A comprehensive review of the matrix metalloproteinase field including a complete bibliography of the literature through of Figure 1. Pathways for degradation of the extracellular matrix. overlap are indicated by boxes. Areas of *Department of Oral Biology and Research Center in Oral Biology, School Dentistry, University of Alabama at Birmingham, Birmingham, AL. 1990 compiled by Woessner1 was recently published.2 For shorter general discussions of the role of MMP in health and disease the reader is referred to recent reviews.3,4 At the outset it is important to recognize that much of the evidence for the role of MMP in specific developmental or pathological processes remains circumstantial and indirect. That is certainly true for the involvement of MMP in human periodontal diseases. It has proven difficult to specifically identify the natural substrates for most MMP in part because of their relatively broad and overlapping substrate specific- Volume 64 Number 5 BIRKEDAL-HANSEN 475 (Table 1). Moreover, the relationship of the MMPdependent pathway to other routes of matrix degradation has not been adequately clarified. Yet the observation that MMP transcripts or protein frequently are present in cells, tissues, and interstitial fluids in vivo strengthens the notion ities that these enzymes are involved in the metabolic remodeling of the extracellular matrix. monocytes/macrophages,14*15 osteoblasts,16*17 and chondrocytes.18 The highly glycosylated PMN enzyme has a considerably larger molecular mass than FIB-CL (Mr 75,000 vs. 57,000/52,000) but the 2 protein cores are of virtually identical size. The 2 collagenases differ markedly in terms of transcriptional regulation. PMN-type collagenase is rapidly released from specific granule storage sites of triggered PMNs whereas FIB-CL is synthesized on demand by initiating transcription of the corresponding gene. This process causes a lag period of 6 to 12 hours before enzyme can be detected in the extracellular environment. The stromelysin group of MMP includes SL-1 and SL-2 which have virtually identical substrate specificities and cleave a wide range of extracellular matrix proteins (proteoglycan core protein, type IV and V collagen, fibronectin, and laminin.)19 SL-1 is expressed by stromal cells either constitutively or after induction by growth factors/cytokines [ILl,20 EGF,21 TNF-a,20 PDGF21] or phorbol esters.22*23 The enzyme does not appear to be expressed by PMN leukocytes and keratinocytes in the human, but a SL-1 (or SL-2) homologue is induced in murine skin epidermis by treatment with the tumor promoter TPA (12-O-tetradecanoyl-phorbol acetate).23*24 SL-2 is expressed less abundantly than SL-1 and does not appear to respond to growth factors (EGF and IL- Matrix Metalloproteinases The MMP gene family encodes 9 or more metal-dependent endopeptidases which collectively are capable of degrading most, if not all, extracellular matrix macromolecules (Table 1). The enzymes share extensive sequence homology but differ somewhat in terms of substrate specificity and transcriptional regulation. All MMP may be regarded as derivatives of a 5-domain prototype structure, formed either by addition or deletion of regulatory domains as shown in Figure 2. A short signal sequence is followed in order by a propeptide which endows the virgin enzyme with catalytic latency, a catalytic domain which contains the active site and the catalytic machinery, a proline-rich hinge region, and a pexin-like COOH-terminal domain which plays a role in determining substrate specificity. In addition, the 2 gelatinases contain a gelatin-binding insert (fibronectin type II-like repeats) in the catalytic domain.5*6 Each of the enzymes contains a tridentate Zn++-binding site believed to constitute the active site.7 The pexin-like domain is absent from the smallest MMP, PUMP-1. The PMN-CL gene is only expressed by PMN leukocytes whereas the highly homologous FIB-CL gene is expressed by fibroblasts,8*9 keratinocytes,10,11 endothelial cells,12*13 Table 1: Matrix The Mr 72K GL is perhaps the most widely distributed of all MMP and has been identified in fibroblasts,25 keratinocytes,26 endothelial cells,27 monocytes/macrophages,28 osteoblasts,29 and chondrocytes.30 Mr 72K GL does not appear, however, to be expressed by PMN leukocytes but is present in a circulating form in plasma.31 The Mr 92K 1) or phorbol esters. Metalloproteinase Family Abbreviation FIB-CL PMN-CL Enzyme Interstitial Collagenases Fibroblast-type Collagenase PMN-type Collagenase Stromelysins Stromelysin-1 Stomelysin-2 Gelatinases MMP# MMP-1 Mr Extracellular Matrix Substrates 57,000/52,000 Collagen I, II, III, VII, VIII, X, Gelatin Same as FIB-CL MMP-8 75,000 60,000/55,000 SL-1 SL-2 MMP-3 MMP-10 MMP-2 PG Core Protein, Fibronectin, Laminin, Collagen IV, V, IX, X, Elastin as 60,000/55,000 72,000 92,000 n.d. Same SL-1 Mr 72K Gelatinase/ Mr 72K GL Type IV Collagenase Mr 92K Gelatinase/ Mr 92K GL Type IV Collagenase MMP-9 Gelatin, Collagen IV, V, VII, X, Elastin, Fibronectin Gelatin, Collagen IV, V, Elastin n.d. Other PUMP-1 Stromelysin-3 Metallo- SL-3 PUMP-1 MME MMP-11 MMP-7 28,000 55,000 Fibronectin, Laminin, Collagen IV, Gelatin, proCL, PG Core Protein Elastin elastase MMP Macrophage numbering according to Nagase et al.13 J Periodontol 476 MATRIX METALLOPROTEINASES HINGE PROPEPTIDE CATALYTIC DOMAIN PEXIN-LIKE DOMAIN , May 1993 (Supplement) Cytokines on Reference Table 2. Transcriptional Effect of Growth Factors and Matrix Metalloproteinases Induction PROTOTYPE FIBRONECTIN-LIKE INSERT PROLINE-HICH HINGE Reference Mr 72 K GL, Mr 92 K GL IL-1 a,ß TNF-a TGF-a EGF PDGF bFGF NGF 20, 45 20, 46, 47 49 21 Repression TGF-ß IFN-7 50, 52 53, 54 21, 48 44, 50 51 SL-1,-2,-3 PMN-CL, FIB-CL TGF-ß 26, 41 PUMP-1 Propeptido Domain structure Figure 2. of matrix metalloproteinases. GL is produced by PMN leukocytes,32'33 keratinocytes,26'34 and monocytes/macrophages,35 and occasionally by fibroblasts. It is interesting to note that PMN which express a unique and distinct CL gene utilize the same Mr 92K GL gene as other cells although in a manner which yields a storable rather than a secreted enzyme. Mr 72K GL is expressed constitutively by most cells in culture and responds only moderately to growth factors which induce the Mr 92K GL. The Mr 72K and Mr 92K GL cleave a number of peptide bonds in denatured collagen chains to yield small peptides.25'36 Besides gelatin, gelatinases also cleave types IV, V, VII, and X collagens37"39 and elastin.40 PUMP-1 cleaves a wide range of substrates including fibronectin, laminin, and gelatin. The enzyme is expressed in gingival fibroblasts41 and has been isolated from the involuting rat uterus and from rectal carcinoma cells.42,43 SL-3 transcripts are expressed by mesenchymal cells of human mammary carcinomas, often adjacent to invading epithelial tumor cells. SL-3 expression is also induced in embryonic fibroblasts by several growth factors. It is unknown whether SL-3 is produced by cells of human periodontal tissues. The amino acid sequence deduced from cDNA suggests that SL-3 encodes a functional metalloproteinase, but the enzyme protein has not yet been isolated or characterized. SL-3 has the same principal domain structure as CLs and SL-1 and SL-2, but differs by an insert of 10 residues at the autolytic activation site.44 LATENT ACTIVE Figure 3. Activation of MMP precursors cysteine switch. is initiated by opening of the Regulation of MMP Activity The activity of MMP against extracellular matrix substrates is regulated at 4 "gates": 1) by transcriptional regulation of MMP genes; 2) by precursor activation; 3) by differences in substrate specificity; and 4) by MMP inhibitors. Transcriptional regulation. The transcription of most MMP genes is regulated by endogenous growth factors and cytokines (Table 2). Stimulation or repression of growth factor- and cytokine-responsive MMP genes results in up to a 50-fold change in mRNA and protein levels. Transcription of the CL and SL-1 genes (and in some cells the SL-3 and Mr 92K GL genes as well) is induced by IL- action that falls in this category is the induction of FIB-CL in macrophages by LPS.60'61 In addition, microbial proteinases62,63 and lectins64 may also play a role in the transcriptional regulation of MMP expression. Activation of precursors. The latency of MMP precursors appears to be maintained, at least in part, by a coordinate bond which links an unpaired Cys residue in the propeptide to the active site Zn++65,66 (Fig. 3). Disruption of the Cys-Zn++ bond is a prerequisite to activation and may be achieved in a number of different ways: 1) by interaction or modification of the Cys residue by organomercurials, metal ions, thiol reagents, and oxidants; 2) by conformational change of the Polypeptide backbone induced by certain chaotropic agents (KI, NaSCN) and detergents (SDS); and 3) by excision of a portion of the propepeptide by proteolytic enzymes (trypsin, plasmin, chymotrypsin, neutrophil elastate, cathepsin B, and plasma kallikrein). The enzyme subsequently catalyzes 1 or more autolytic cleavages to generate the fully-processed form.66 69 Iß,20,45 TNF-a,20'46'47 PDGF,21,48 TGF-a,49 EGF,21 bFGF,20,50 NGF,51 and with few exceptions26 abrogated by TGF-ß50,52 and IFN-7.53,54 TGF-ß which ablates transcription of CL and SL-150,52 upregulates Mr 72K GL by 2- to 4-fold and Mr 92K by up to 8-fold.26,41 These findings are of considerable interest because TGF-ß in general appears to down-regulate rather than stimulate MMP expression. Transcription of MMP genes may also be regulated by other endogenous pathways. FIB-CL is induced or stimulated in osteoblasts by PTH and 1,25 di(OH)D3,17,55'56 and FIB-CL and SL-1 are down-regulated by glucocorticoids and by retinoids.57 59 The proximity of a viable microbial plaque, moreover, creates an unequaled opportunity for direct transcriptional effects by microbial metabolites on human gingival and periodontal cells. A potentially important inter- Volume 64 Number 5 BIRKEDAL-HANSEN 477 CL and SL-1, at least in cell culture systems, and that SL1 is perhaps involved in the processing of the FIB-CL precursor to a particularly highly-active form.70-72 There is still insufficient evidence to determine whether these reactions also take place in vivo. It is of note, however, that the degradation of gelatin and collagen by PMN leukocytes73'74 involves activation of CL and Mr 92K GL by oxidative pathways, presumably by oxidation of the unpaired propeptide Cys residue by HOC1. Substrate Specificity. A certain level of regulation of MMP activity is encoded at the level of the substrate. Although the enzymes have somewhat overlapping substrate specificities, there are also notable differences, particularly with respect to the cleavage of collagens.2 Virtually all of the enzymes cleave gelatin and fibronectin at some rate, and most cleave type IV and V collagens at sufficiently high temperatures. The characteristic ability of FIB-CL and PMN-type collagenases to dissolve interstitial collagen fibrils by cleavage of the component molecules in the proteinase-resistant triple-helical domain, however, is not shared by other members of the family. Inhibition Although MMP can readily be activated in the test tube by any or all of these mechanisms, the biologic activation mechanisms are still poorly understood. Several studies have suggested that plasmin may play a role in activation of FIB- perhaps by other cells as well.13,92,93 The inhibitor forms a complex with the zymogen form of Mr 72K GL.5,89 TIMP2 is 2- to 10-fold more effective than TIMP-1 against the 2 GL whereas TIMP-1 appears to more effectively inhibit CL.91 The 2 TIMP genes are differently regulated. TIMP1 expression is stimulated by growth factors (EGF, TNFa, IL-1, TGF-ß), phorbol esters, retinoids, and glucocorticoids, whereas TIMP-2 expression is down-regulated by TGF-ß and fails to respond to phorbol esters.41,94 98 ROLE OF MMP IN HUMAN PERIODONTAL DISEASES of MMP by Different Cell Types of Human Periodontal Tissues Each of the major cell types of human periodontal tissues is capable of expressing a unique complement of MMP when properly stimulated as summarized in Table 3. PMN leukocytes share with many other cell types the ability to express the common 92K GL gene but, for reasons that are unknown, utilize a distinct gene for interstitial collagenase (PMN-CL). Both enzymes are stored in specific granules and rapidly released when the cells are triggered.99 Fibroblasts express a somewhat larger complement of MMP, Expression captured by a-macroglobulins by unique venus-fly-trap mechanism activated by cleavage of a bond in the "bait region."75,76 This cleavage leads to hydrolysis of a labile internal thiol-ester bond and covalent cross-linking of a nascent glytamyl residue to lysyl side chains exposed on the surface of the attacking proteinase.77 The rapid capture rates, particularly with CL, suggest that a-macroglobulins, particularly alM, play an important role in the regulation of MMP activity. are a a-Macroglobulins. Active MMP including an interstitial, fibroblast-type collagenase (FIBCL), SL-1, SL-3, Mr 72K GL, and PUMP-1, but not Mr 92K GL. Keratinocytes express FIB-CL, Mr 72K GL, and Mr 92 K GL, but not SL-1. Macrophages and endothelial cells each express FIB-CL, the Mr 92K GL.12,13,20,39 SL-1, and both the Mr 72K and functional inhibitor can be recovered from the complex.90 TIMP-1 also forms a complex with the zymogen form of Mr 92K GL.5,79,91 TIMP-2, a Mr 22,000 unglycosylated protein, is expressed by fibroblasts and endothelial cells and cluding fibroblasts,86 keratinocytes,87 monocytes/macrophages,15 and endothelial cells.13 TIMP-1 is a Mr 28K glycosylated protein with a Mr 20K protein core.80,86 TIMP-1 forms complexes with active collagenase, but not procollagenase, in a 1:1 stoichiometry with rCjS of 10~9M to 10" 10M.88,89 Complex formation with active MMP does not appear to depend on cleavage of the inhibitor and fully Tissue inhibitors of metalloproteinases (TIMPs). TIMPS form classical non-covalent bimolecular complexes with the active forms of MMPs and, in some instances, with latent MMP precursors as well. TIMPs appear to regulate matrix degradation both by proteinase elimination and by blockage of autolytic MMP activation.78,79 Two members of the TIMP family, TIMP-1 and TIMP-2, have been identified but more may exist.13,80"83 TIMPs are widely distributed in tissues and fluids84,85 and are expressed by many cell types in- Cell Type-Specific Differences in Regulation of MMP The synthesis and packaging of MMP in specific granules are essentially complete when the PMN enters the blood stream. The triggered release of MMP from specific granules is clearly not a transcriptional event and it is still uncertain to what extent it is regulated by growth factors and cytokines. The response pattern of the PMN leukocyte is clearly unique and different from that of any other cell type not only in the complement of MMP expressed but also in the utilization of the enzymes. The PMN leukocyte has evolved to respond rapidly and in full force to external stimuli. No other cell type is capable of "dumping" such large quantities of destructive enzymes in a matter of minutes. The response is sustained, when needed, by continuous recruitment of new cells and not by prolonged release from cells already triggered. Fibroblasts, keratinocytes, macrophages, and endothelial cells share an essentially common response to catabolic growth factors such as IL1, TNF-a, TGF-a, EGF, bFGF, PDGF, namely induction or stimulation of transcription of MMP genes. However, there are important cell-specific and cytokine-specific differences among these responses. In fibroblasts, IL-lß induces primarily SL-1, TNF-a primarily CL,20 IFN-7 represses SL-1 but moderately upregulates CL,53 TGF-ß represses CL and SL-1, but upregulates 72K GL. In kera- 478 MATRIX METALLOPROTEINASES Table 3. Tissues May Expression of Matrix J Periodontol 1993 (Supplement) Metalloproteinases by Five Major Cell Types Fibroblast of Human Periodontal PMNCell Type Enzymes expressed Transcriptional activation Leukocyte PMN-CL, Mr 92K GL Keratinocyte FIB-CL, Mr 72 K GL, Mr 92K GL, SL-2 Macrophage FIB-CL, SL-1, Mr 72K GL, Mr 92K GL TPA Endothelial Cell FIB-CL, SL-1, Mr 7K GL, PUMP-1, SL-3 FIB-CL, SL-1, Mr 72K GL, Mr 92K GL IL-1, TNF-a, TNF-a, EGF, EGF, TGF-a, TGF-a, PDGF, TPA TGF-ß, TPA TPA Mobilization of enzymes Granule Release Seconds Minutes Transcriptional activation 6 to 12 hours Transcriptional activation 6 to 12 hours Transcriptional activation 6 to 12 hours Transcriptional activation 6 to 12 hours Response time Response duration Days Days attachment loss. Recent studies have started to shed light into the mechanisms that enable growth factors and cytokines to elicit overlapping, yet occasionally distinct, transcriptional effects. The AP-1 binding site is a necessary, but not sufficient, requirement for transcriptional activation of MMP expression by many growth factors and cytokines. For instance, the Mr 92K GL gene which contains 3 copies of the AP-1 site is inducible by catabolic growth factors whereas the Mr 72K GL gene which contains no such elements is not.100-101 The transcriptional activity of the AP-1 site depends on binding of c-fos/c-jun dimers to the TGAGTCA sequence. The role of fos, however, is quite complex and even among transcriptional pathways that operate through the AP-1 site, some but not all, are c-fos-dependent. For instance, induction of SL-1 by PDGF is fos-dependent whereas that of EGF is not.21 Moreover, abrogation of FIBCL and SL-1 transcription by TGF-ß is also dependent on c-fos although the action of this growth factor is indepen- tinocytes, TGF-ß upregulates both the Mr 72 K and the Mr 92K GL;26 on the other hand, IL-lß, which is the most potent known inducer of CL- and SL-l-expression by fibroblasts, has no effect on keratinocytes, at least not in the human.11 Recent studies in our own laboratory have shown that growth factors and cytokines which induce expression of SL-1 in stromal cells induce exclusively SL-2 in keratinocytes (unpublished data). The findings summarized above show: 1) that different cell types express different complements of MMP; 2) different cytokines elicit different transcriptional effects in the same cell type; and 3) that different cell types do not necessarily respond in the same fashion to a given cytokine. In the light of these findings, and in the light of the many potential synergistic and antagonistic interactions that exist between 2 or more cytokines, it is apparent that different inflammatory infiltrates may vary considerably in destructive potential. This is perhaps the key to understanding why inflammation of the human gingiva may or may not give rise to tissue destruction and dent of the AP-1 site and instead identified promoter and enhancer elements required for maximal transcriptional activation that are specific for each MMP. 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- bp TGF-ß-responsive element.50,52 Recent studies have depends on a unique 10 moter.102"104 The second messenger-signaling pathways which mediate growth factor and cytokine transcriptional effects on MMP gene expression are still incompletely understood. A body of evidence suggests that protein kinase C (PKC), a cellular receptor for TPA, acts as a messenger in the transcriptional regulation of growth factor-responsive MMP genes in some but not all cases.53,105 cAMP may be involved as a second messenger in some responses but apparently through activation of a different (Jun B-dependent) transcriptional machinery than either TPA or growth factors/cytokines.58,106 cAMP stimulates MMP expression in some cell types; in others leads to repression. For instance, expression of CL and SL-1 by guinea pig peritoneal macrophages, rat UMR-106 osteosarcoma cells, and rat fibroblasts is stimulated by cAMP,24,56,107 whereas the constitutive expression of CL by human GM637 fibroblasts,104 and the IL-1, EGF- and oncogene-induced transcription of FIB-CL and SL-1 genes by rat and human fibroblasts is repressed.52,108 What Are the Important Cytokines and Growth Factors? The complexity of the cytokine networks and the many different cellular responses among resident and immigrant cells of inflamed periodontal tissues give rise to an almost unlimited number of putative pathogenic processes. Clearly not all of these are equally important, but it is not yet possible to identify those cellular interactions that actually drive attachment loss. It is likely that IL-lß plays an important role in regulating MMP expression in inflamed human gingiva. IL-la and -ß are present in concentrations of 10"9-10_8M109,110 which are clearly sufficient to induce Volume 64 Number 5 Table 4. Tissue Destructive BIRKEDAL-HANSEN 479 Enzymes Fluid and Inhibitors in Inflammatory Exudates, Saliva, Concentration and Plasma Enzyme/Inhibitor Collagenase GCF M-g/ml 1-10 Reference 115, Estimate 127 (PMN) Mr 92K Mr 72K gelatinase gelatinase Saliva Plasma GCF GCF Plasma 0.340 0.5-0.6 0.1-0.5 40-200 31 Estimate Plasminogen TIMP Estimate 140 128 Estimate 143 Synovial Plasma GCF fluid 200 0.3-4.6 1.0-1.5 0.2-1.0 700-1100 141, 142 a2-Macroglobulin Synovial GCF Plasma fluid 200-2,400 2,250 143 120, 121, 122 expression of SL-1 and CL in cultured fibroblasts. (10~91010 M). IL-1 is also a potent inducer of bone resorption, but it is not known whether this response is related to the inductive effect of IL-1 on MMP. TNF-a is also present in inducing concentrations in the range 0.1-13 nM.111 It is reasonable to assume that TGF-a may also be involved, although the concentration of TGF-a in GCF has not yet been determined. TGF-a is a potent inducer of CL and Mr 92K GL in keratinocytes, and recent studies have shown that the EGF-receptor (which also serves as a receptor for TGF-a) is expressed abundantly on epithelial cells of the oral mucosa.112 MMP Are Expressed by Gingival/Periodontal Cells In Vivo Woolley and Davis113 investigated the expression of MMP in normal or inflamed human gingiva by immunolocalization methods. FIB-CL was associated with cells in the gingival connective tissue often in close proximity of the junctional and pocket epithelia but not in any epithelial cells. Enzyme was also associated with collagen fibrils and oc- casionally with inflammatory infiltrates. Our own studies have shown that only low levels of MMP are expressed in any one section. Cells scattered throughout the connective tissue (fibroblasts and/or macrophages) show staining of the perinuclear region with antibodies to FIB-CL. Mr 72K GL is not infrequently associated with blood vessels and capillaries. Even heavily-inflamed human gingiva shows only sporadic expression of MMP with no discernible pattern of distribution (unpublished data). In the aggregate, immunolocalization studies have proven fairly inconclusive and have given few, if any clues, to the level of involvement of MMP in periodontal diseases. MMP in GCF and Saliva GCF and saliva contain at least 2 MMP, CL, and Mr 92K GL.114"116 CL appears to be exclusively or predominantly of the PMN-type.117,118 The apparent absence of SL-1 and Mr 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 PMN leukocytes are the dominant, and perhaps the only significant, contributors of MMP in GCF. If so, the question arises whether the enzymes are indeed released from PMNs in the tissue or the crevicular fluid, or perhaps still inside the cells at the time of sampling and only secondarily released as a sampling artifact. "GCF enzymes" produced by PMN leukocytes and perhaps carried to the GCF still inside the cells include CL, Mr 92K GL, elastase, and myeloperoxidase. Measurement of these activities in oral fluid samples appears to be essentially an enzyme-based PMN count, a concern previously voiced by Cimasoni.119 Metabolites in GCF may originate from 3 different compartments: from plasma, from resident cells/stroma, and from PMN leukocytes (Table 4). Serum components including a-2M, albumin, and Igs typically are present at high concentrations, varying between 20% and 100% of plasma values. a2-M concentrations, for instance, vary between 0.2-2.4 mg/ml or 10 to 1000 times higher than any of the MMP.120123 Because of the high concentration of a2-M, it is unlikely that any MMP exists in GCF in an active form. This notion apparently conflicts with the finding of more active (versus latent) MMP in Periodontitis patients/sites,123 126 but as previously alluded to it is uncertain 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 not been accurately determined, we predict (based on the concentration of other plasma proteins) that it is as high as 40 to 200 |xg/ml. By comparison, we have previously estimated that the concentration of CL may reach 10 |xg/ml, but it is probably much lower. Since Mr 72K GL is present in plasma at a concentration of approximately 0.5 (xg/ml,31 we also predict that this enzyme, although undetectable by zymographic analysis, exists in GCF at a concentration of 0.1 to 0.5 |xg/ml even in the absence of any local contribution from the tissues. Mr 92K GL, presumably of PMN 480 MATRIX METALLOPROTEINASES J Periodontol May REFERENCES 1. Woessner JF, Jr. Literature 1993 (Supplement) is present in saliva in relatively high levels (340 ng/ ml).127 It is also readily detectable in GCF112 but the concentration is not known. TIMP-1 (or TIMP-2) levels in GCF are unknown but based on the plasma concentration (1.01.5 u,g/ml)128 we estimate that TIMP concentrations of GCF are at least 0.2 to 1.0 Lig/ml. Metabolites that originate from the resident cell/stroma presumably are washed away by the outward fluid flow provided they escape local capture and elimination. It is likely the PGE2, IL-1, and TNF-a are true metabolites released from cells indigenous to the area or perhaps newlyrecruited macrophages. Recent studies have shown, however, that triggered PMN leukocytes activate transcription of the IL-1 gene.129 It therefore cannot be excluded that PMN leukocytes are a major source of IL-1 in GCF. origin, 2. 3. 4. 5. 6. Association of GCF MMP With Disease Severity A number of studies have aimed at correlating MMP activities with disease severity. Several experiments have shown that when group means are compared, CL activity increases with disease severity (Periodontitis > gingivitis > health) and that treatment tends to return GCF values to control levels.115'124-126 It has not been possible to convincingly demonstrate similar correlations for individual sites, presumably because of the high site-to-site, person-to-person, and day-to-day variation. Neither the "level" nor the "pattern" of activity at the site seems to correlate well with "bone loss" over a 6-month period.130,131 When monitoring a particular site over an extended period of time, the high degree of week-to-week variation suggests that a single measurement taken at a particular point in time has little, if any, predictive value. Sporadic "high" values never remain high for long and do not necessarily herald impending bone loss.130,131 Role of MMP in Bone Resorption A number of studies have suggested that osteoblasts express FIB-CL when stimulated by bone-resorbing agents.16,56 These observations have led to the hypothesis that osteoclastic bone resorption is initiated by an Osteoblast response to resorptive signals such as PTH, which includes expression of FIB-CL and perhaps other MMP, and result in dissolution of the unmineralized collagenous osteoid layer.132 Osteoblasts later vacate the surface as newly-recruited osteoclasts move in. Osteoclasts do not appear to express MMP, but utilize a distinct acidic cathepsin-dependent mechanism for dissolution of mineralized matrices.133'134 It is possible, although not yet proven, that expression of MMP is an early key event in bone resorption, a finding that may help explain why bone resorption is so highly sensitive to IL-1 and TNF-a.135138 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Acknowledgments This study was supported by DE06028. 19. NIH grants DE08228 and 20. on vertebrate matrix metalloproteinases and their inhibitors. In: Birkedal-Hansen H, Werb Z, Welgus HG, Van Hart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:425-501. Birkedal-Hansen H, Werb Z, Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992. Woessner JF Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991; 5:2145-2154. Birkedal-Hansen H. 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