Birkedal 1993

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. Matrix Metalloproteinases. A Review. In: Alvarez OA, ed. Critical Reviews in Oral Biology and Medicine. Boca Raton, FL: CRC Press; 1993; 4:197-250. Goldberg Gl, Marmer BL, Grant GA, Eisen AZ, Wilhelm S, He C. Human 72-kilodalton type IV collagenase forms a complex with a tissue inhibitor of metalloproteases designated TIMP-2. Proc Natl Acad Sei (USA) 1989; 86:8207-8211. Collier IE, Krasnov PA, Strongin YA, Birkedal-Hansen H, Goldberg Gl. Alanine scanning mutagenesis and functional analysis of the fibronectin-like collagen binding domain from human 92kDa type IV collagenase. / Biol Chem 1992; 267:6776-6781. Vallee BL, Auld DS. Active zinc binding sites of zinc metalloenzymes. In: Birkedal-Hansen H, Werb Z, Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:5-19. Goldberg Gl, Wilhelm SM, Kronberger A, Bauer EA, Grant GE, Eisen AZ. Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein. JBiol Chem 1986; 261:6600-6605. Stricklin GP, Bauer EA, Jeffrey JJ, Eisen AZ. Human skin collagenase: Isolation of precursor and active forms from both fibroblast and organ cultures. Biochemistry 1977; 16:1607-1615. Lin HY, Wells BR, Taylor RE, Birkedal-Hansen H. Degradation of type I collagen by rat mucosal keratinocytes. / Biol Chem 1987; 262:6823-6831. Petersen MJ, Woodley DT, Stricklin GP, O'Keefe EJ. Production of procollagenase by cultured human keratinocytes. / Biol Chem 1987; 262:835-840. Moscatelli D, Jaffe E, Rifkin DB. Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells. Cell 1980; 20:343-351. Herron GS, Banda MJ, Clark EJ, Gavrilovic J, and Werb Z. Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. J Biol Chem 1986; 261:2814-2818. Welgus HG, Campbell EJ, Bar-Shavit Z, Senior RM, Teitelbaum SL. Human alveolar macrophages produce a fibroblast-like collagenase and collagenase inhibitor. J Clin Invest 1985; 76:219-224. Campbell EJ, Cury JD, Lazarus CJ, Welgus HG. Monocyte procollagenase and tissue inhibitor of metalloproteinases. / Biol Chem 1987; 262:15862-15868. Otsuka K, Sodek J, Limeback H. Synthesis of collagenase and collagenase inhibitors by osteoblast-like cells in culture. EurJ Biochem 1984; 145:123-129. Quinn CO, Scott DK, Brinckerhoff CE, Matrisian LM, Jeffrey JJ, Partridge NC. Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells. / Biol Chem 1990; 265:22342-22347. Lefebvre V, Peeters-Joris C, Vaes G. Modulation by interleukin-1 and tumor necrosis factor a of production of collagenase, tissue inhibitor of metalloproteinases and collagen types in differentiated and dedifferentiated articular chondrocytes. Biochim Biophys Acta 1990; 1052:366-378. Nicholson R, Murphy G, Breathnach R. Human and rat malignanttumor-associated mRNAs encode stromelysin-like metalloproteinases. Biochemistry 1989; 28:5195-5203. MacNaul KL, Chartrain N, Lark M, Tocci MJ, Hutchinson NI. Volume 64 Number 5 BIRKEDAL-HANSEN 39. 481 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. Discoordinate expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases-1 in rheumatoid human synovial fibroblasts. JBiol Chem 1990; 265:17238-17245. Kerr LD, Holt JT, Matrisian LM. Growth factors regulate transin gene expression by c-/os-dependent and o/os-independent pathways. Science 1988; 242:1424-1427. Chin JR, Murphy G, Werb Z. Stromelysin, a connective tissuedegrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. / Biol Chem 1985; 260:12367-12376. Wilhelm SM, Collier IE, Kronberger A, et al. Human skin fibroblast stromelysin: Structure, glycosylation substrate specificity, and differential expression in normal and tumorigenic cells. Proc NatlAcad Sei (USA) 1987; 84:6725-6729. Matrisian LM, Bowden GT, Krieg P, et al. The mRNA coding for the secreted protease transin is expressed more abundantly in malignant than in benign tumors. Proc NatlAcad Sei (USA) 1986; 83:94139417. Seltzer JL, Adams SA, Grant GA, Eisen AZ. Purification and properties of a gelatin-specific neutral protease from human skin. / Biol Chem 1981; 256:4662-4668. Salo T, Lyons JG, Rahemtulla F, Birkedal-Hansen H, Larjava H. Transforming growth factor-ßl up-regulates type IV collagenase expression in cultured human keratinocytes. / Biol Chem 1991; 266:11436-11441. Kalebic T, Garbisa S, Glaser B, Liotta LA. Basement membrane collagen: Degradation by migrating endothelial cells. Science 1983; 221:281-283. Garbisa S, Ballin M, Daga-Gordini D, et al. Transient expression of type IV collagenolytic metalloproteinase by human mononuclear phagocytes. / Biol Chem 1986; 261:2369-2375. Overall CM, Sodek J. Initial characterization of a neutral metalloproteinase, active on native 3/4-colIagen fragments, synthesized by ROS 17/2.8 osteoblastic cells, periodontal fibroblasts, and identified in gingival crevicular fluid. J Dent Res 1987; 66:1271-1282. Lefebvre V, Peeters-Joris C, Vaes G. Production of gelatin-degrading matrix metalloproteinases ("type IV collagenases") and inhibitors by articular chondrocytes during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor a. Biochim Biophys Acta 1991; 1094:8-18. Zucker S, Lysik RM, Gurfinkel MH, et al. Immunoassays of type IV collagenase/gelatinase (MMP-2) in human plasma. ImmunolMeth 1992; 148:189-198. Hibbs MS, Hasty KA, Seyer JM, Kang AH, Mainardi CM. Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase. / Biol Chem 1985; 260:24932500. Murphy G, Ward R, Hembry RM, Reynolds JJ, Kuhn K, Tryggvason K. Characterization of gelatinase from pig polymorphonuclear leukocytes. Biochem J 1989; 258:463^172. Wilhelm SM, Collier IE, Manner BL, Eisen AZ, Grant GA, Goldberg GI. SV40-transformed human lung fibroblasts secrete a 92kDa type IV collagenase which is identical to that secreted by normal human macrophages. / Biol Chem 1989; 264:17213-17221. Mainardi CL, Hibbs MS, Hasty KA, Seyer JM. Purification of a type IV collagen degrading metalloproteinase from rabbit alveolar macrophages. Coll Relat Res 1984; 4:479-492. Seltzer JL, Akers KT, Weingarten H, Grant GA, McCourt DW, Eisen AZ. Cleavage specificity of human skin type IV collagenase (gelatinase). JBiol Chem 1990; 265:20409-20413. Seltzer JL, Eisen AZ, Bauer EA, Morris NP, Glanville RW, Burgeson RE. Cleavage of type VII collagen by interstitial collagenase and type IV collagenase (gelatinase) derived from human skin. / Biol Chem 1989; 264:3822-3826. Gadher SJ, Schmid TM, Heck LW, Woolley DE. Cleavage of collagen type X by human synovial collagenase and neutrophil elastase. Matrix 1989; 9:109-115. Biol Chem 1990; 265:13521-13527. 40. Senior R, Griffin G, Fliszar CJ, Shapiro SD, Goldberg GI, Welgus HG. Human 92- and 72-kilodalton type IV collagenases are elastases. JBiol Chem 1991; 266:7870-7875. 41. Overall CM, Wrana JL, Sodek J. Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-ßl in human fibroblasts. J Biol Chem 1991; 266:14064-14071. 42. Woessner JF, Taplin CJ. Purification and properties of a small latent matrix metalloproteinase of the rat uterus. / Biol Chem 1988; 263:16918-16925. 43. Miyazaki K, Hattori Y, Umenishi F, Yasumitsu H, Umeda M. Purification and characterization of extracellular matrix-degrading metalloproteinase, matrin (Pump-1) secreted from human rectal carcinoma cell line. Cancer Res 1990; 50:7758-7764. 44. Basset P, Bellocq JP, Wolf C, et al. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinoma. Nature 1990; 348:699-704. 45. Wahl L, Wahl SM, Mergenhagen SE, Martin GR. Collagenase production by endotoxin-activated macrophages. Proc Natl Acad Sei (USA) 1974; 71:3598-3601. 46. Uitto V-J, Larjava H, Heino J, Sorsa T. A protease from Bacteroides gingivalis degrades cell surface and matrix glycoproteins of cultured gingival fibroblasts and induces secretion of collagenase and Plasminogen activator. Infect Immun 1989; 57:213-218. 47. Birkedal-Hansen H, Wells BR, Lin H-Y, Caufield PW, Taylor RE. Activation of keratinocyte-mediated collagen (type I) breakdown by suspected human periodontopathogen: Evidence of a novel mechanism of connective tissue breakdown. J Periodont Res 1984; 19:645650. 48. Bauer EA, Cooper TW, Huang JS, Altman J, Deuel TF. Stimulation of in vitro human skin collagenase expression by platelet-derived growth factor. Proc NatlAcad Sei (USA) 1985; 82:4132-4136. 49. Lyons JG, Lin H-Y, Salo T, et al. Expression of collagen-cleaving matrix metalloproteinases by keratinocytes. Effect of growth factors and cytokines and of microbial mediators. In: Hamada S, Holt SC, McGhee JR, eds. Periodontal Disease: Pathogens and Host Immune Responses. Tokyo: Quintessence Publishing Co.; 1991:291-305. 50. Edwards DR, Murphy G, Reynolds JJ, et al. Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 1987; 6:1899-1904. 51. Machida CM, Scott JD, Ciment G. NGF-induction of the metalloproteinase transin/stromelysin in PC 12 cells: Involvement of multiple protein kinases. / Cell Biol 1991; 114:1037-1048. 52. Kerr LD, Miller DB, Matrisian LM. TFG-ßl Inhibition of transin/ stromelysin gene expression is mediated through a Fos binding sequence. Cell 1990; 61:267-278. 53. Unemori EN, Bair MJ, Bauer EA, Amento EP. Stromelysin expression regulates collagenase activation in human fibroblasts. Dissociable control of two metalloproteinases by interferon-7. J Biol Chem 1991; 266:23477-23482. 54. Andrews HJ, Bunning RAD, Plumpton TA, Clark IM, Russell RGG, Cawston TE. Inhibition of interleukin-l-induced collagenase production in human articular chondrocytes in vitro by recombinant human interferon-gamma. Arthritis Rheum 1990; 33:1733-1738. 55. Delaisse J-M, Eeckhout Y, Vaes G. Bone-resorbing agents affect the production and distribution of procollagenase as well as the activity of collagenase in bone tissue. Endocrinology 1988; 123:264276. 56. Civitelli R, Hruska KA, Jeffrey JJ, Kahn AJ, Avioli LV, Partridge NC. Second messenger signaling in the regulation of collagenase production by osteogenic sarcoma cells Endocrinology 1989; 124:2928-2934. 57. Jonat C, Rahmsdorf HJ, Park KK, et al. Antitumor promotion and Welgus HG, Fliszar a, Seltzer JL, Schmid TM, Jeffrey JJ. Differential susceptibility of type X collagen to cleavage by two mammalian interstitial collagenases and 72kDa type IV collagenase. / 482 MATRIX METALLOPROTEINASES May J Periodontol 1993 (Supplement) antiinflammation: Down-modulation of AP-1 (Fos/Jun) activity by Cell 1990; 62:1189-1204. 58. Nicholson RC, Mader S, Nagpal S, Leid M, Rochette-Egly C, Chambon P. Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP-1 binding site. EMBO J 1990; 9:4443^1454. 59. Frisch SM, Ruley HE. Transcription from the stromelysin promoter is induced by Interleukin-1 and repressed by dexamethasone. / Biol Chem 1987; 262:16300-16304. 60. Wahl L, Wahl SM, Mergenhagen SE, Martin GE. Collagenase production by endotoxin-activated macrophages. Proc Natl Acad Sei (USA) 1974; 71:3598-3601. 61. Cury J, Campbell EJ, Lazarus CJ, Albin RJ, Welgus HG. Selective upregulation of human alveolar macrophage collagenase production by lipopolysaccharide and comparison to collagenase production by fibroblasts. / Immunol 1988; 141:4306-4312. 62. Dayer JM, Beutler B, Cerami A. Cachectin/tumor necrosis factor stimulates collagenase and Prostaglandin E2 production by human synovial cells and dermal fibroblasts. J Exp Med 1985; 162:21632168. 63. Brenner DA, O'Hara M, Angel P, Chojkier M, Karin M. Prolonged activation of jun and collagenase genes by tumour necrosis factora. Nature 1989; 337:661-663. 64. Overall CM, Sodek J. Concanavalin A produces a matrix-degradative phenotype in human fibroblasts. J Biol Chem 1990; 265:2114121151. 65. Springman EB, Angleton EL, Birkedal-Hansen H, Van Wart HE. Multiple modes of activation of latent human fibroblast collagenase: Evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation. Proc Natt Acad Sei (USA) 1990; 87:364-368. 66. Van Wart HE, Birkedal-Hansen H. The cysteine switch: A principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc Natl Acad Sei (USA) 1990; 87:5578-5582. 67. Grant GA, Eisen AZ, Marmer BL, Roswit WT, Goldberg GI. The activation of human skin fibroblast procollagenase. J Biol Chem 1987; 262:5886-5889. 68. Nagase H, Enghild JJ, Suzuki K, Salvesen G. Stepwise activation of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate. Biochemistry 1990; 29:5783-5789. 69. Suzuki K, Enghild JJ, Morodomi T, Salvesen G. The activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry 1990; 29:10261-10270. 70. Mignatti P, Robbins E, Rifkin DB. Tumor invasion through the human amniotic membrane: requirement for a Proteinase cascade. Cell 1986; 47:487^198. 71. He C, Wilhelm SM, Pentland AP, et al. Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc Natl Acad Sei (USA) 1989; 86:2632-2636. 72. Birkedal-Hansen H, Lin H-Y, Birkedal-Hansen B, Windsor LT, Pierson MC. Degradation of collagen fibrils by live cells. Role of expression and activation of procollagenase. In: Birkedal-Hansen H, Werb Z, Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:368-374. 73. Peppin GJ, Weiss SJ. Activation of the endogenous metalloproteinase, gelatinase, by triggered human neutrophils. Proc Natl Acad Sei (USA) 1986; 83:4322^1326. 74. Weiss SJ, Peppin G, Ortiz X, Ragsdale C, Test ST. Oxidative autoactivation of latent collagenase by human neutrophils. Science 1985; 227:747-749. 75. Sottrup-Jensen L. Structure, shape, and mechanism of Proteinase complex formation. J Biol Chem 1989; 264:11539-11542. 76. Sottrup-Jensen L, Sand O, Kristensen L, Fey GH. The ot-macroglobulin bait region. Sequence diversity and localization of cleavage glucocorticoid hormone. sites for proteinases in five mammalian a-macroglobulins. J Biol Chem 1989; 264:15781-15789. 77. Sottrup-Jensen L, Hansen HF, Christensen U. Generation and reactivity of "nascent" a2-macroglobulin: Localization of cross-links in a2-macroglobulin-trypsin complex. Ann NY Acad Sei 1983; 421:188208. 78. DeClerck YA, Yean TD, Lu HS, Ting J, Langley KE. Inhibition of autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MI/TIMP-2. / Biol Chem 1991; 266:3893-3899. 79. Howard EW, Bullen EC, Banda MJ. Regulation of the autoactivation of human 72-kDa progelatinase by tissue inhibitor of metalloproteinases-2. J Biol Chem 1991; 266:13064-13069. 80. Carmichael DF, Sommer A, Thompson RC, et al. Primary structure and cDNA cloning of human fibroblast collagenase inhibitor. Proc Natl Acad Sei (USA) 1986; 83:2407-2411. 81. Docherty AJP, Lyons A, Smith BJ, et al. Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature 1985; 318:66-69. 82. Stetler-Stevenson WG, Krutzsch HC, Liotta LA. Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. J Biol Chem 1989; 264:17374-17378. 83. Boone TC, Johnson MJ, DeClerck YA, Langley KE. cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases. Proc Natl Acad Sei (USA) 1990; 87:2800-2804. 84. Murphy G, Docherty AJP, Hembry RM, Reynolds JJ. Metalloproteinases and tissue damage. BrJ Rheumatol 1991; 30:25-31. 85. Welgus HG, Bauer EA, Stricklin GP. Elevated levels of human collagenase inhibitor in blister fluids of diverse etiology. J Invest Dermatol 1986; 258:12252-12258. 86. Stricklin GP, Welgus HG. Human skin fibroblast collagenase inhibitor. Purification and biochemical characterization. / Biol Chem 1983; 258:12252-12258. 87. Lin H-Y. Production of Collagenase by Normal Human Keratinocytes. [Thesis]. Birmingham, Alabama: University of Alabama at Birmingham, 1987:19-20. 88. Cawston TE, Murphy G, Mercer E, Galloway WA, Hazleman BL, Reynolds JJ. The interaction of purified rabbit bone collagenase with purified rabbit bone metalloproteinase inhibitors. Biochem J 1983; 211:313-318. 89. Welgus HG, Jeffrey JJ, Eisen AZ, Roswit WT, Stricklin GP. Human skin fibroblast collagenase: Interaction with substrate and inhibitor. Coll Relat Res 1985; 5:167-179. 90. Murphy G, Koklitis P, Carne AF. Dissociation of tissue inhibitor of metalloproteinases (TIMP) from enzyme complexes yields fully active inhibitor. Biochem J 1989; 261:1031-1034. 91. Howard EW, Bullen EC, Banda MJ. Preferential inhibition of 72 and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2. JBiol Chem 1991; 266:13070-13075. 92. DeClerck YA, Yean TD, Ratzkin BJ, Lu HS, Langley KE. Purification and characterization of two related but distinct metalloproteinase inhibitors secreted by bovine aortic endothelial cells. / Biol Chem 1989; 264:17445-17453. 93. Stetler-Stevenson WG, Krutzsch HC, Liotta LA. Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. JBiol Chem 1989; 264:17374-17378. 94. Stetler-Stevenson WG, Brown PD, Onisto M, Levy AT, Liotta LA. Tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression in tumor cell lines and human tumor tissues. J Biol Chem 1990; 265:13933-13938. 95. Clark SD, Kobayashi DK, Welgus HG. Regulation of the expression of tissue inhibitor of metalloproteinases and collagenase by retinoids and glucocorticoids in human fibroblasts. J Clin Invest 1987; 80:12801288. 96. Coulombe B, Skup D. In vitro synthesis of the active tissue inhibitor Volume 64 Number 5_BIRKEDAL-HANSEN 116. Lee W, Aitken 483 of metalloproteinases encoded by a complementary DNA from virusinfected murine fibroblasts. J Biol Chem 1988; 263:1439-1443. 97. Nomura S, Hogan BLM, Wills AJ, Heath JK, Edwards DR. Developmental expression of tissue inhibitor of metalloproteinases (TIMP) RNA. Development 1989; 105:575-584. 98. Mawatari M, Kohno K, Mizoguchi H, et al. Effects of tumor necrosis factor and epidermal growth factor on cell surface morphology, cell surface receptors, and the production of tissue inhibitor of metalloproteinases and IL-6 in human microvascular endothelial cells. J Immunol 1989; 143:1619-1627. 99. Hibbs MS, Bainton DF. Human neutrophil gelatinase is a component of specific granules. / Clin Invest 1989; 84:1395-1402. 100. Huhtala P, Chow LT, Tryggvason K. Structure of the human type IV collagenase gene. J Biol Chem 1990; 265:11077-11082. 101. Huhtala P, Tuuttila A, Chow LT, Lohi J, Keski-Oja J, Tryggvason K. Complete structure of the human gene for 92-kDa type IV collagenase. Divergent regulation of expression for the 92- and 72kilodalton enzyme genes in HT-1080 cells. / Biol Chem 1991; 266:16485-16490. 102. Auble DT, Brinckerhoff CE. The AP-1 sequence is necessary but not sufficient for phorbol induction of collagenase in fibroblasts. Biochemistry 1991; 30:4629-4635. 103. Sirum-Connolly K, Brinckerhoff CE. Interleukin-1 or phorbol induction of the stromelysin promoter requires an element that cooperates with AP-1. Nucleic Acids Res 1991; 19:335-341. 104. Gutman A, Wasylyk B. The collagenase gene promoter contains a TPA and oncogene-responsive unit encompassing the PEA3 and AP1 binding sites. EMBO J 1990; 9:2241-2246. 105. McDonnell SE, Kerr LD, Matrisian LM. Epidermal growth factor stimulation of stromelysin mRNA in rat fibroblasts requires induction of proto-oncogenes c-fos and c-jun and activation of protein kinase C. Mol Cell Biol 1990; 10:4284-4293. 106. Angel P, Karin M. Specific members of the Jun protein family regulate collagenase expression in response to various extracellular stimuli. In: Birkedal-Hansen H, Werb Z, Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:156-164. 107. McCarthy JB, Wahl SM, Rees J, Olsen CE, Sandberg AL, Wahl LM. Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate. / Immunol 1980; 124:2405-2409. 108. Takahashi S, Ito A, Nagino Y, Mori Y, Xie B, Nagase H. Cyclic adenosine 3',5'-monophosphate suppresses interleukin-1-induced synthesis of matrix metalloproteinases but not tissue inhibitor of metalloproteinases in human uterine cervical fibroblasts. J Biol Chem 1991; 166:19894-19899. 109. Masada MP, Persson R, Kenney JS, Lee SW, Page RC, Allison AC. Measurement of interleukin-la and -Iß in gingival crevicular fluid: Implications for the pathogenesis or periodontal disease. / Periodont Res 1990; 25:156-163. 110. Offenbacher S, Heasman PA, Collins JG. Modulation of host PGE2 secretion as a determinant of periodontal disease expression. J Periodont 1993; 64(suppl.):432-444. 111. Rossomando EF, White LB. A novel method for the detection of TNF-alpha in gingival crevicular fluid. / Periodontal 1993; 64(suppl): 445^149. 112. Nordlund L, Hormia M, Saxen L, Thesleff I. Immunolohistochemical localization of epidermal growth factor receptors in human gingival epithelia. J Periodont Res 1991; 26:333-338. 113. Woolley DE, Davies RM. Immunolocalization of collagenase in periodontal tissues. / Periodont Res 1981; 16:292-297. 114. Gangbar S, Overall CM, McCulloch AG, Sodek J. Identification of polymorphonuclear leukocyte collagenase and gelatinase activities in mouthrinse samples: Correlation with periodontal disease activity in adult and juvenile Periodontitis. J Periodont Res 1990; 25:257267. 115. Villela B, Cogen RB, Bartolucci AA, Birkedal-Hansen H. Collagenolytic activity in crevicular fluid from patients with chronic adult Periodontitis, localized juvenile Periodontitis and gingivitis, and from healthy control subjects. J Periodont Res 1987; 22:381-389. 117. S, Kulkarni G, et al. Collagenase activity in recurrent Periodontitis; Relationship to disease progression and doxycycline therapy. J Periodont Res 1991; 26:479-485. Sorsa T, Uitto V-J, Suomolainen M, Vauhkonen M, Lindy S. Comparison of interstitial collagenases from human gingiva, sulcular fluid and polymorphonuclear leukocytes. J Periodont Res 1988; 23:386- 393. 118. Uitto V-J, Suomolainen K, Sorsa T. Salivary collagenase. Origin, characteristics and relationship to periodontal health. J Periodont Res 1990; 25:135-142. 119. Cimasoni G. Crevicular Fluid Updated. In: Myers HM, ed. Monographs in Oral Science, vol. 12. Basel: S. Karger; 1983. 120. Schenkein HA, Genco RJ. Gingival fluid and serum in periodontal diseases. I. Quantitative study of Immunoglobulins, complement components, and other plasma proteins. J Periodontol 1977; 48:772- 777. 121. Tollefsen T, Saltvedt E. Comparative analysis of gingival fluid and plasma by crossed immunoelectrophoresis. J Periodont Res 1980; 15:96-106. 122. Condacci I, Cimasoni G, Ahmad-Zadeh C. a2-macroglobulin in sulci from healthy and inflamed human gingivae. Infect Immun 1982; 36:66-71. 123. Sengupta S, Lamster IB, Knocht A, Duffy TA, Gordon JM. The effect of treatment on IgG, IgA, IgM and a-2-macroglobulin in gingival crevicular fluid from patients with chronic adult Periodontitis. Arch Oral Biol 1988; 33:425-431. 124. Kryshtalskyj E, Sodek J. Nature of collagenolytic enzyme and inhibitor in crevicular fluid from healthy and inflamed periodontal tissues of beagle dogs. / Periodont Res 1987; 22:264-269. 125. Kryshtalskyj E, Sodek J, Ferrier JM. Correlation of collagenolytic enzymes and inhibitors in gingival crevicular fluid with clinical and microscopic changes in experimental Periodontitis. Arch Oral Biol 1986; 31:21-31. 126. Larivee J, Sodek J, Ferrier JM. Collagenase and collagenase inhibitor activities in crevicular fluid of patients receiving treatment for localized juvenile Periodontitis. J Periodont Res 1986; 21:702715. 127. Davis GA. Identification of an abundant latent 94kDa gelatin-degrading metalloproteinase in human saliva which is activated by acid exposure: Implications for a role in digestion of collagenous proteins. Arch Biochem Biophys 1991; 286:551-554. 128. Cawston T, McLaughlin P, Coughlan R, Kyle V, Hazleman B. Synovial fluids from infected joints contain metalloproteinase-tissue inhibitor of metalloproteinase (TIMP) complexes. Biochim Biophys Acta 1990; 1033:96-102. 129. Marucha PT, Zeff RA, Kreutzer DL. Regulation of IL-lß gene expression in human peripheral blood PMN. / Periodont Res 1991; 26:264-267. 130. Cogen RB, Heaven T, Jeffcoat M, Birkedal-Hansen H. Periodontal disease activity determined by digital subtraction radiography. / Dent Res 1989; 68(spec. issue):(Abstr. 111). 131. Birkedal-Hansen H, Pierson M, Heaven T, Jeffcoat M, Cogen RB. Bone loss, GCF collagenase and GCF flow in human Periodontitis. J Dent Res 1989; 68(spec. issue):(Abstr. 1221). 132. Vaes G, Delaisse J-M, Eeckhout Y. Relative roles of collagenase and lysosomal cysteine-proteinases in bone resorption. In: BirkedalHansen H, Werb Z, Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:383-388. 133. Blair HC, Kahn AJ, Crouch EC, Jeffrey JJ, Teitelbaum SL. Isolated osteoclasts resorb the organic and inorganic components of bone. / Cell Biol 1986; 102:1164-1172. 134. Vaes G. Cellular biology and biochemical mechanism of bone resorption. Clin Orthop 1988; 231:239-271. 135. Gowen M, Mundy GR. Actions of recombinant interleukin 1, in- J Periodontol 484 MATRIX METALLOPROTEINASES May 1993 (Supplement) 141. 136. 137. 138. 139. 140. terleukin 2, and interferon--y on bone resorption in vitro. / Immunol 1986; 136:2478-2482. Bertolini DR, Nedwin GE, Bringman TS, Smith DD, Mundy GR. Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors. Nature 1986; 319:516-518. Saklatvala J. Tumour necrosis factor a stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature 1986; 322:547-549. Heath JK, Saklatvala J, Meikle MC, Atkinson SJ, Reynolds JJ. Pig interleukin-1 (catabolin) is a potent stimulator of bone resorption in vitro. Calcif Tissue Int 1985; 37:95-97. Nagase H, Barrett AJ, Woessner JF, Jr. Nomenclature and glossary of the matrix metalloproteinases. In: Birkedal-Hansen H, Werb Z, Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:421-424. Deutsch DG, Mertz ET. Plasminogen: Purification from human plasma by affinity chromatography. Science 1970; 170:1095-1096. vial fluid of human rheumatoid arthritis. Biomed Res 1991; 12:169— 173. 142. Cawston TE, McLaughlin P, Hazleman BL. Paired serum and synovial fluid values of a2-macroglobulin and TTMP in rheumatoid arthritis. BrJRheumatol 1987; 26:354-358. 143. Virca GD, Mallya RK, Pepys MB, Schnebli HP. Quantitation of human leukocyte elastase, cathepsin G, ct-2-macroglobulin and ct1-proteinase inhibitor in Osteoarthritis and rheumatoid arthritis synovial fluids. Adv Exp Med Biol 1982; 167:345-353. Send reprint requests to: Dr. Henning Birkedal-Hansen, Department of Oral Biology, University of Alabama School of Dentistry, University of Alabama at Birmingham, UAB Station, Birmingham, AL 35294. Hayakawa T, Yamashita K, Kodama S, Iwata H, Iwata K. Tissue inhibitor of metalloproteinases and collagenase activity in syno-