Gingival Crevicular Fluid MatrixMetalloproteinase (MMP)-7, ExtracellularMMP Inducer, and Tissue Inhibitor ofMMP-1 Levels in Periodontal DiseaseGulnur Emingil,* Taina Tervahartiala,†‡ Paivi Mantyla,†‡ Marko Maatta,§i Timo Sorsa,†‡

and Gul Atilla*

Background: During periodontal inflammation, matrix metalloproteinases (MMPs) are under the controlof several regulatory mechanisms including the upregulation of expression by inducers and downregulationby inhibitors. Our study aimed to examine the levels and molecular forms of MMP-7, tissue inhibitor of MMP(TIMP)-1, and extracellular matrix metalloproteinase inducer (EMMPRIN) in gingival crevicular fluid (GCF)from patients with different periodontal diseases.

Methods: A total of 80 subjects (20 patients with generalized aggressive periodontitis [GAgP], 20 withchronic periodontitis [CP], 20 with gingivitis, and 20 periodontally healthy subjects) were included in thisstudy. Periodontal status was evaluated by measuring probing depth, clinical attachment loss, presenceof bleeding on probing, and plaque. GCF MMP-7, TIMP-1, and EMMPRIN levels and molecular formswere analyzed by enzyme-linked immunosorbent assay (ELISA) and Western immunoblot techniques us-ing specific antibodies.

Results: Total amounts of GCF MMP-7 were found to be similar between the study groups. GAgP, CP, andgingivitis groups had significantly higher total amounts of GCF EMMPRIN compared to healthy subjects (P<0.008). Among the patient groups, the GAgP group had the highest total amount of GCF EMMPRIN relativeto the gingivitis group (P = 0.0004). Soluble EMMPRIN existed in GCF in multiple molecular-weight speciesespecially in periodontitis-affected GCF under non-reducing conditions, i.e., 30-, 55-, 100-, 180-, and 200-kDa species. All patient groups had significantly elevated total amounts of GCF TIMP-1 relative to thehealthy group (P <0.0001). GAgP and CP groups also had a higher total amount of GCF TIMP-1 comparedto the gingivitis group (P <0.0001 and P <0.0001, respectively). The GAgP group had higher GCF TIMP-1and EMMPRIN levels compared to the CP group, but this elevation did not reach statistical significance.

Conclusions: Our data indicate that MMP-7 is associated with the innate host defense in periodontal tis-sues. Increased EMMPRIN and TIMP-1 levels in GCF are associated with the enhanced severity of periodon-tal inflammation, indicating that these molecules can participate in the regulation of progression ofperiodontal diseases. To our knowledge, the present study demonstrated the presence of soluble formsof EMMPRIN in GCF of patients with different periodontal diseases for the first time. J Periodontol2006;77:2040-2050.

KEY WORDS

Enzyme-linked immunosorbent assay; gingival crevicular fluid; matrix metalloproteinase;periodontal diseases/pathogenesis; Western immunoblotting.

* Department of Periodontology, School of Dentistry, Ege University, _IIzmir, Turkey.† Institute of Dentistry, University of Helsinki, Helsinki, Finland.‡ Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland.§ Department of Ophthalmology, Helsinki University Central Hospital.i Department of Pathology, University of Oulu, Oulu, Finland.

doi: 10.1902/jop.2006.060144

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The integrity of the extracellular matrix andbasement membrane is important in maintain-ing the stability of periodontal tissues.1-3 Tissue

integrity is maintained by a balance between matrixdegradation and production, which is regulated to alarge extent through the action of matrix metal-loproteinases (MMPs) in both normal tissue remod-eling and pathological states.4,5 MMPs belong to thematrixin superfamily, which is composed of at least24 genetically distinct but structurally related zinc-containing enzymes that differ in their molecular struc-ture, substrate specificity, and cellular origin. MMPsare implicated in a variety of tissue-destructive patho-logical conditions, including periodontitis, physiolog-ical processes, such as tissue remodeling associatedwith tooth development and eruption as well as woundhealing, and anti-inflammatory defensive processes.3-7

MMPs can be classified into at least five subgroupsincludingcollagenases,gelatinases,stromelysin,mem-brane-type collagenases, and matrilysins accordingto their structural and functional characteristics.8

Within the MMP family, matrilysin-1/MMP-7 is thesmallest of the known MMPs.9 MMP-7 is unique bythe absence of the hemopexin domain that is commonto all other MMPs.9,10 This enables pro-MMP-7 to bindtissue inhibitors of MMPs (TIMPs), whereas activatedMMP-7 can bind to TIMPs but less sensitively.10 Unlikeother MMPs, which are produced and released onlyupon response to injury, MMP-7 is mainly producedin many non-injured exocrine and mucosal epitheliarather than connective tissue cells.11,12 MMP-7 hasa capacity to degrade numerous extracellular matrixand basement membrane components includingfibronectin, laminin, entactin, and elastin and non-extracellular matrix proteins such as a1-proteinaseinhibitor.13-17 In contrast, triple-helical native intersti-tial collagen is resistant to proteolytic cleavage byMMP-7, but MMP-7 can activate pro-MMP-8.18 In ad-dition to its activity on extracellular matrix proteins, itcan also cleave a variety of secreted and cell surfaceproteins.9 MMP-7 contributes to the innate hostdefense by converting antimicrobial prodefensin pep-tides to their active forms and, therefore, is associatedwith the mucosal antimicrobial defense. The presenceof MMP-7 expression has been demonstrated in thesuprabasal cells of the junctional epithelium, whereasthere is no MMP-7 expression in inflamed connectivetissues and in the pocket epithelium.19 MMP-7 haspreviously been shown to be present in peri-implantsulcular fluid (PISF) and expressed in gingival tissuesof periodontitis patients.20,21

The proteolytic activity of MMPs can be regulatedby changes in the delicate balance between the ex-pression and synthesis of MMPs and TIMPs.3,22 Evi-dence has shown that tissue destruction in the

disease process might result from MMP/TIMP imbal-ance in the diseased tissues.23-25 TIMPs are expressedby the cells that also produce MMPs such as fibro-blasts, keratinocytes, monocyte/macrophages, andendothelial cells.22,23 TIMPs, which consist of fourmembers, TIMP-1, -2, -3, and -4, have many basicsimilarities, but they exhibit structural and biochemi-cal differences.22 Among TIMPs, TIMP-1 was demon-strated to be the major inhibitor of MMPs in gingivaltissues of patients with periodontal disease.24-26 How-ever, previous studies demonstrated controversialresults about TIMP-1 levels in periodontal disease.Both increased24-30 and decreased31-33 TIMP-1 levelswere reported in gingival crevicular fluid (GCF) andgingival tissues of patients with periodontitis.

MMP activity is under control of several regulatorymechanisms including the upregulation of expressionby inducers.34,35 Extracellular matrix metalloprotein-ase inducer (EMMPRIN) or CD147 is a highly glyco-sylated plasma membrane bound glycoprotein of 4555 kDa. It contains two immunoglobulin superfamilydomains, a transmembrane and a stoplasmic do-main.36 EMMPRIN was originally identified on the sur-face of tumor cells, and its expression on tumor cellsmay induce tumor progression and invasion by trig-gering the production or release of MMPs by fibro-blasts and endothelial cells.37-40 EMMPRIN can alsoplay roles in cell attachment, cell migration, and cell-to-cell interactions. It was demonstrated that EMM-PRIN can stimulate MMP-1, -2, and -3 production,whereas it shows no effect on TIMPs.37,38,41,42 There-fore, EMMPRIN could promote the collagenolytic bal-ance in favor of MMP production and activation. Itsprecise molecular function is not completely under-stood, but EMMPRIN has also been identified in manydiseased human tissues such as rheumatoid arthritisand in normal keratinocytes.34,43,44 The ability ofEMMPRIN to stimulate MMP production suggests thatthis molecule could be related to several normal andpathological tissue modulatory processes and tissueremodeling.34,37 To date, it is not known if this novelmolecule is involved in the regulation of immune re-sponses in periodontal tissues. Therefore, the aim ofthe present study was to investigate, for the first timeto our knowledge, the presence of levels and molecu-lar forms of EMMPRIN together with MMP-7 and TIMP-1levels in gingival crevicular fluid (GCF) samples ofpatients with different periodontal diseases and to testwhether crevicular levels of these molecules correlatewith each other and with clinical parameters.

MATERIALS AND METHODS

Study PopulationA total of 80 subjects were included in this study. Allconsecutive subjects were recruited from the Depart-ment of Periodontology, School of Dentistry, Ege

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University, over a period of 2 years between 2002 and2004.Thestudywasconducted inaccordancewith theHelsinki Declaration of 1975, as revised in 2000. Thepurposeof thestudywascompletelyexplained toeachsubject before they entered the study, and informedconsent was obtained from each subject. Completemedical and dental histories were taken from all sub-jects. All of the patients were non-smokers and hadat least 20 teeth. None of the subjects had a historyof systemicdiseaseorhad receivedantibioticsorothermedications or periodontal treatment within the past 4months.Patientswithseveremedicaldisorders includ-ing diabetes mellitus, immunological disorders, andpregnant females were excluded from the study. Theselection of the patients was made according to theclinical and radiographic criteria proposed by the1999 International World Workshop for a Classifica-tion of Periodontal Disease and Conditions.45

Generalized aggressive periodontitis group (GAgP).The GAgP group included nine females and 11 malesranging in age from 18 and 39 years (mean: 29.4 – 6.4years). These patients demonstrated a generalizedpattern of severe destruction and clinical attachmentloss (CAL) ‡4 mm on eight or more teeth; at leastthree of these teeth were other than central incisorsor first molars.

Chronic periodontitis group (CP). The CP groupconsisted of seven females and 13 males betweenthe ages of 39 and 63 years (mean: 50.9 – 5.7 years).They had moderate to severe alveolar bone loss andCAL ‡5 mm and probing depth (PD) ‡6 mm in multi-ple sites of all four quadrants of the mouth. Diagnosisof CP was made if the CAL was commensurate withthe amount of local factors of the patient.

Gingivitis group. The gingivitis group includedeight females and 12 males with varying degrees ofgingival inflammation, but no signs of attachment losswere observed. Clinical evidence of gingivitis in thesepatients was based on the presence of bleeding onprobing at any site. These patients ranged in age from15 to 58 years (mean: 32.1 – 11.5 years).

Healthy group. Thehealthygroupconsistedof11fe-males and nine males who exhibited PD <3 mm and noCAL,bleedingonprobing,andradiographicevidenceofbone loss. These individuals were healthy volunteersfrom the Department of Periodontology, who rangedin age from 20 to 50 years (mean: 27.4 – 9.5 years).

Determination of Periodontal StatusAt the screening stage, to determine the clinical peri-odontalstatus,all subjectshadaclinicalperiodontalex-amination including the measurement of PD and CALby one examiner (GE). Dichotomous measurementsof supragingival plaque accumulation and bleedingon probing were also recorded.All measurements wereperformed at six sites per tooth for the whole mouth.

Collection of GCF SamplesAfter being selected for the study, subjects were re-called for GCF sampling. In the GAgP and CP groups,GCF samples were collected from approximal sites oftwo non-adjacent teeth with ‡6 mm PD. In the gingivi-tis group, GCF sampling was done from approximalsites of two non-adjacent teeth with bleeding and ‡2mm PD. In the healthy group, GCF samples were col-lected from approximal sites of two non-adjacentteeth with £2 mm PD. Prior to GCF sampling, the sup-ragingival plaque was removed from the interproxi-mal surfaces with a sterile curet; these surfaces weredried gently by an air syringe and were isolated by cot-ton rolls. GCF was sampled with filter paper. Paperstrips were carefully inserted into the crevice until mildresistance was felt and left there for 30 seconds.46

Care was taken to avoid mechanical injury. Stripscontaminated with blood were discarded.47 The ab-sorbed GCF volume of each strip was determinedby electronic impedance¶ and placed into a sterileEppendorf vials and kept at -40�C until analyzed.The readings from the electronic gingival fluid mea-suring device# were converted to an actual volume(ml) by reference to the standard curve.

GCF ProcessingThe absorbed fluid was eluted from each strip into 75ml 0.2 M NaCl, 1 mM CaCl2, 50 mM Tris-HCl, pH 7.8,for 2 hours at 22�C, and 14 ml of this eluate/extractwas boiled in 5 ml 4· Laemli-sample buffer (no reduc-tants) for 3 minutes at 100�C. After vortexing, the19-ml mixture was applied to each gel slot.

Analysis of MMP-7 and TIMP-1 by Enzyme-LinkedImmunosorbent Assay (ELISA)ELISA for MMP-7 and TIMP-1. MMP-7 and TIMP-1levels in the studied GCF groups were determined byELISA assays** according to the manufacturer’s in-structions. GCF samples were assayed at dilutions 1:10and1:15forMMP-7andTIMP-1,respectively.TheELISAforMMP-7detectscomplexed,proform,active,andfrag-mentedspeciesofMMP-7.TheELISAforTIMP-1detectsnative, complexed, and fragmented species of TIMP-1.The detection limits for total MMP-7 and TIMP-1 byELISA are 0.097 and 1.25 ng/ml, respectively.

Analysis of EMMPRINWestern immunoblotting was used to detect the levelsand molecular forms of EMMPRIN in 160 GCF sam-ples. Western immunoblotting was performed usingthe monoclonal antibody specific for EMMPRIN2 asdescribed previously.48,49 Human recombinant EMM-PRIN/Fc/His (NSO) was used as a positive control and

¶ Periotron 8000, ProFlow, Amityville, NY.# Periotron 8000, ProFlow.** R&D Systems, Amersham, Little Chanfolt, U.K.

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was kindly obtained as a prerelease reagent from themanufacturer.†† The protocol of the Western blottinganalysis system‡‡ was followed according to the man-ufacturer’s instructions. A total of 1 mM b-mercapto-ethanol was used when reduction was needed. Thedetection limit by EMMPRIN Western immunoblottingis ;1 ng/lane under non-reducing and reducing condi-tions. Data are expressed as densitometric units (du).49

Total MMP-7, EMMPRIN, and TIMP-1 data were de-termined by averaging the two single sites per subject.The calculation of the concentration data for each en-zyme was performed by dividing the amount of themediator by the volume of the sample.

Statistical AnalysisStatistical analysis was performed using non-para-metrical techniques. The subject was used as the unitof measurement. Comparisons between the studygroups were performed using the Kruskal-Wallis test.When there were significant differences (P <0.05),post-hoc two-group comparisons were assessed withBonferroni-corrected Mann-Whitney U tests, and Pvalues <0.008 were considered to be statistically sig-nificant. Spearman rank correlation analysis was usedto analyze the correlations between GCF MMP-7,EMMPRIN, and TIMP-1 levels and clinical parameters,and P <0.05 was considered significant. All data anal-ysis was performed using a statistical package.§§

RESULTS

Clinical FindingsThe mean clinical data for the sampling areas areshown in Table 1.PD and CAL. As expected, the GAgP and CP groupshad significantly higher mean PD scores compared tothe healthy group (P <0.0001 and P <0.0001, respec-tively). These groups also had significantly highermean PD scores compared to the gingivitis group(P <0.0001 and P <0.0001, respectively). The meanCALs of sampling sites in GAgP and CP groups weresignificantly higher than the healthy group (P <0.0001and P <0.0001, respectively). These groups also hadsignificantly higher mean CAL than the gingivitisgroup (P <0.0001 and P <0.0001, respectively). TheGAgP and CP groups had similar PD and CAL scores(P = 0.3752 and P = 0.4623, respectively).

Percentage of sites with bleeding on probing andplaque. The GAgP, CP, and gingivitis groups had asignificantly higher percentage of sites with bleed-ing on probing compared to the healthy group (P<0.0001, P <0.0001, and P <0.0001, respectively).They had significantly higher percentage of sites withplaque compared to the healthy group (P = 0.0004, P =0.0002, and P <0.0001, respectively). The GAgP, CP,and gingivitis groups had a similar percentage of siteswith bleeding (P = 0.3418 and P = 0.0754, respec-

tively). The percentages of sites with plaque were alsonot different between GAgP, CP, and gingivitis groups(P = 0.9289 and P = 0.2694, respectively).

GCF volume. The GAgP and CP groups had a sig-nificantly higher mean GCF volume compared to thehealthy group (P <0.0001 and P <0.0001, respec-tively). These groups also had a significantly highermean GCF volume compared to the gingivitis group(P <0.0001 and P <0.0001, respectively). The GAgPand CP groups had a similar mean GCF volume (P =0.3103). The gingivitis group had a significantly highermean GCF volume than that of the healthy group(P <0.0001).

Biochemical FindingsGCF MMP-7 levels. The distribution of the totalamount of GCF MMP-7 is shown in Figure 1. Therewas not any significant difference in the total amountof GCF MMP-7 between study groups (P = 0.0931)(Table 2 and Fig. 1).

When the data were expressed as concentrations,significant differences were found between studygroups (P <0.0001). The GAgP, CP, and gingivitisgroups had lower GCF MMP-7 concentrations com-pared to that of healthy group (P <0.0001, P <0.0001,and P = 0.0004, respectively). The GCF MMP-7 concen-tration of the gingivitis group was significantly higherthan that of GAgP and CP groups (P <0.0001 andP <0.0001, respectively) (Table 3). No significant differ-ences were found between GAgP and CP groups (P =0.7660).

GCF EMMPRIN levels. The distribution of totalamounts of GCF EMMPRIN of the study groups aregiven in Figure 2. GAgP, CP, and gingivitis groupshad significantly higher total amount of GCF EMM-PRIN compared to healthy subjects (P <0.0001,P <0.0001, and P <0.0001, respectively). Among thepatient groups, the GAgP group had the highest totalamount of GCF EMMPRIN compared to the gingivitisgroup (P = 0.0004). The total amount of GCF EMM-PRIN of the CP group was similar to that of the gingi-vitis group (P = 0.7251). GAgP patients had a highertotal amount of GCF EMMPRIN compared to the CPgroup, but this did not reach significance (P =0.0389) (Table 2 and Fig. 2).

When the data were expressed as concentration,GCF EMMPRIN concentration of the GAgP and gingi-vitis groups were significantly higher compared tothose of the healthy group (P = 0.0001 and P <0.0001,respectively). The CP group tended to have a higherconcentration of GCF EMMPRIN compared to thehealthy group, although this difference did not achievestatistical significance,basedoncorrection formultiple

†† AF 972, R&D Systems.‡‡ ECL, Amersham Pharmacia Biotech, Buckinghamshire, U.K.§§ Abacus Concepts, Berkeley, CA.

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testing (P = 0.0398). The GCF EMMPRIN concentra-tion of GAgP was not different from the concentrationsof CP and gingivitis groups (P = 0.0403 and P =0.9776, respectively). There was also not any signifi-cantdifferenceinGCFEMMPRINconcentrationbetweenthe CP and gingivitis groups (P = 0.0173) (Table 2).

Existence of soluble EMMPRIN in the GCFsamples. Western immunoblotting was performedfirst without any reducing agent (b-mercaptoethanol)(Fig. 3, lanes 1 through 8) to detect the molecularforms of soluble EMMPRIN occurring naturally in theGCF samples; one common feature was that severalsoluble immunoreactive EMMPRIN species could beseen (Fig. 3, lanes 1 through 8). The immunoreactiveEMMPRIN species of 30, 55, and 100 to 200 kDacould be detected in GCFs of these 55 kDa; 100 to200 kDa was associated especially with periodontitis;180 to 200 with gingivitis; and very low, hardly detect-able EMMPRIN immunoreactive species were found in

healthy GCF, respectively. Slightly elevated levels of20- to 30-kDa EMMPRIN species could be detected inperiodontitis GCF relative to gingivitis and healthyGCF (Fig. 3, lanes 1 through 8). The next 1 mMb-mercaptoethanol was used as a reducing agent,and the Western immunoblotting was repeated. The

Table 1.

Clinical Parameters of the Sampling Areas in Study Groups (mean – SD)

GAgP CP Gingivitis Healthy P Value

PD (mm) 8.0 – 1.7* 7.6 – 1.5* 2.6 – 0.5† 1.9 – 0.8 <0.0001‡

CAL (mm) 8.4 – 1.7* 8.7 – 1.8* 0 0 <0.0001‡

Sites with bleeding on probing (%) 92.5 – 18.3† 80 – 37.7† 100† 0 <0.0001‡

Sites with plaque (%) 80 – 37.7† 82.5 – 33.5† 90 – 30.8† 0 <0.0001‡

GCF (ml) 0.57 – 0.2* 0.50 – 0.2* 0.27 – 0.1† 0.15 – 0.1 <0.0001‡

* Significant difference from gingivitis and healthy groups (Mann-Whitney U test, P <0.008).† Significant difference from healthy group (Mann-Whitney U test, P <0.008).‡ Significant difference among groups by Kruskal-Wallis test.

Figure 1.GCF MMP-7 levels of CP, GAgP, gingivitis, and healthy groups.Box plots show medians and 25th and 75th percentiles as boxesand 10th and 90th percentiles as whiskers. Outside values areshown as open circles.

Table 2.

Total Amounts of GCF MMP-7, EMMPRIN,and TIMP-1 of GAgP, CP, Gingivitis, andHealthy Groups

GAgP CP Gingivitis Healthy P Value

MMP-7 (pg/site)

Mean 43 37.33 36.68 36.81

SD 17.1 7.5 8.5 6.8

Median 36.63 34.77 32.50 33.78 0.0931

Minimum 30.63 31.50 30.25 31.12

Maximum 91.58 58.09 61.61 52.26

EMMPRIN (du/site)

Mean 2.20 1.20 0.95 0.19

SD 1.4 0.9 0.6 0.2

Median 1.92 1.02 0.70* 0.12† <0.0001‡

Minimum 0.76 0.09 0.33 0

Maximum 5.47 3.10 2.37 0.72

TIMP-1 (pg/site)

Mean 609 278.57 73.99 46.2

SD 645.2 205.4 17.0 10.2

Median 387.75 237.0 68.25§ 44.63† <0.0001‡

Minimum 87 57 52.5 33.0

Maximum 2676.75 807 115.5 66.75

* Significant difference from GAgP group (Mann-Whitney U test, P <0.008).† Significant difference from GAgP, CP, and gingivitis groups (Mann-

Whitney U test, P <0.008).‡ Significant difference among groups by Kruskal-Wallis test.§ Significant difference from GAgP and CP groups (Mann-Whitney U test,

P <0.008).

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typical feature at this point was that all groups of GCFsamples investigated expressed the monomeric solu-ble 55- to 80-kDa forms of EMMPRIN derived fromhigher molecular-size 100- to 200-kDa EMMPRINspecies (data not shown) similar to human recombi-nant EMMPRIN used as a positive control (Fig. 3, lanes9 and 10).

GCF TIMP-1 levels. Distribution of the total amountof GCF TIMP-1 is shown in Figure 4. Significant differ-ences were found between study groups. The GAgP,CP, and gingivitis groups had a significantly elevatedtotal amount of GCF TIMP-1 compared to the healthygroup (P <0.0001, P <0.0001, and P <0.0001, respec-tively). GAgP and CP groups also had a higher total

Table 3.

GCF MMP-7, EMMPRIN, and TIMP-1Concentration of GAgP, CP, Gingivitis,and Healthy Groups

GAgP CP Gingivitis Healthy P Value

MMP-7 (pg/ml)

Mean 84.0 83.7 157.3 314.6

SD 45.9 40.1 80.8 191.4

Median 71.7* 78.66* 132† 235.49 <0.0001‡

Minimum 34.03 36.52 75.12 84.96

Maximum 227.53 217.16 426.12 757.76

EMMPRIN (du/ml)

Mean 4.2 2.3 3.7 1.3

SD 3.3 1.6 2.2 1.2

Median 2.94† 1.99 2.86† 0.97 <0.0001‡

Minimum 0.76 0.19 1.25 0

Maximum 12.74 6.55 8.4 4.74

TIMP-1 (pg/ml)

Mean 1019.4 559.4 316.5 373.1

SD 786.9 327.9 153.2 204.4

Median 931.74§ 551.55 279.24 304.92 0.0052‡

Minimum 87 140.96 138.32 172.85

Maximum 2676.75 1311.96 754.55 1063.64

* Significant difference from gingivitis and healthy groups (Mann-WhitneyU test, P <0.008).

† Significant difference from healthy group (Mann-Whitney U test,P <0.008).

‡ Significant difference among groups by Kruskal-Wallis test.§ Significant difference from gingivitis group (Mann-Whitney U test,

P <0.008).

Figure 2.GCF EMMPRIN levels of GAgP, CP, gingivitis, and healthy groups.Box plots show medians and 25th and 75th percentiles as boxesand 10th and 90th percentiles as whiskers. Outside values areshown as open circles. *Significantly different from GAgP, CP, andgingivitis groups (Kruskal-Wallis test, P <0.001; Mann-WhitneyU test, P <0.008). †Significantly different from GAgP group(Kruskal-Wallis test, P <0.001; Mann-Whitney U test, P <0.008).

Figure 3.Western immunoblot of EMMPRIN in GCF. Two representativeGCF samples from GAgP, CP, gingivitis, and healthy groups areshown (without reduction: lanes 1-8). Lanes 9 and 10 representpositive control (recombinant human EMMPRIN) with and withoutreduction, respectively. Mobilities of the molecular weight markersare indicated on the left.

Figure 4.GCF TIMP-1 levels of GAgP, CP, gingivitis, and healthy groups.Box plots show medians and 25th and 75th percentiles as boxesand 10th and 90th percentiles as whiskers. Outside values areshown open circles. *Significantly different from GAgP, CP, andgingivitis groups (Kruskal Wallis test, P <0.001; Mann-WhitneyU test, P <0.008). †Significantly different from GAgP and CP groups(Kruskal Wallis test, P <0.001; Mann-Whitney U test, P <0.008).

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amount of GCF TIMP-1 compared to the gingivitisgroup (P <0.0001 and P <0.0001, respectively). Thetotal amount of GCF TIMP-1 of the GAgP group wasalso higher compared to that of the CP group, butthis did not reach significance (P = 0.0760) (Table 2and Fig. 3).

When the data were expressed as concentration,the TIMP-1 concentration of the GAgP, CP, and gingi-vitis groups was comparable to that of the healthygroup (P = 0.0189, P = 0.0744, and P = 0.2733, re-spectively). The gingivitis groups had a lower TIMP-1 concentration compared to that of the GAgP group(P = 0.0051). The TIMP-1 concentration of the CPgroup was similar to the TIMP-1 concentrations of theGAgP and gingivitis groups (P = 0.1027) (Table 2).

The correlation between GCF MMP-7, EMMPRIN,and TIMP-1 levels and clinical parameters ispresentedin Table 4. There was no correlation between the totalamount of GCF MMP-7 and clinical periodontal pa-rameters (P >0.05). The total amount of GCF EMM-PRIN and TIMP-1 was positively correlated with allclinical periodontal parameters (P <0.05), except fora lack of correlation between GCF TIMP-1 and the per-centage of sites with bleeding on probing. There wasalso a strong correlation between total amounts ofGCF EMMPRIN and TIMP-1 (R = 0.641; P <0.0001).

DISCUSSION

The current understanding about the pathogenesis ofperiodontal disease suggests that it is a multifactorialdisease caused by the induction of the host inflamma-tory response against pathogenic bacteria.50 In thepresent study, we investigated MMP-7, EMMPRIN,and TIMP-1 levels in the GCF of patients with differentperiodontal diseases. To the best of our knowledge,the presence of EMMPRIN in soluble and sheddedforms was demonstrated for the first time in humanperiodontitis-affected GCF relative to the periodontally

healthy control GCF. We also demonstrated that totalamounts of EMMPRIN and TIMP-1 are enhanced inGCF as the severity of periodontal disease increased.On the other hand, total amounts of GCF MMP-7 werefound to be similar between groups studied.

It has been generally accepted that analysis of GCFconstituents can provide information of associationbetween a specific metabolic change and disease sta-tus.51,52 GCF constituents such as MMP-8 can be di-agnostically used to monitor the course, treatment,and medication (any drug that was given to patientsfor the supporting of periodontal treatment) of peri-odontal diseases.53-56 However, one important con-sideration in the analysis of host mediators in GCF isthe method of presenting the data. Some researchershave stated that expression of GCF data as totalamount per standardized sampling time is a more sen-sitive way than reporting them as a concentra-tion.51,57 When the GCF volume of the sample islow, the calculation of concentration data gives rela-tively high values that do not reflect that this mediatoris high in the GCF. Because collecting a standardamount of GCF is essential to express the results asa concentration and GCF volume is very small andit exhibits wide variations, expressing GCF data as to-tal activity is more appropriate than reporting them asa concentration.51,57 In the present study, we col-lected GCF samples for the same length of time andreported the data as the total amount per sample aswell as a concentration. However, our findings showedthat total amounts of MMP-7, EMMPRIN, and TIMP-1 in GCF samples were decisive enough to reveal dif-ferences between study groups.

MMPs have diverse and complex actions in manynormal biologic and pathological processes such asrheumatoid arthritis, cancer progression, and peri-odontal disease.4,6,7 MMP-7 was demonstrated to beexpressed by many mucosal tissues such as skinand salivary glands when exposed to bacteria.11 Ithas key roles in epithelial defense and repair.11,13,14

MMP-7 is involved in innate host defense by activatingthe latent form of defensins in normal, intact epithe-lium, and therefore, it can be associated with the an-timicrobial defense of the junctional epithelium.11 Itwas demonstrated that matrilysin-deficient mutantmice exhibit a decreased capacity for antimicrobialdefense in the small intestine.58 Uitto et al.19 showedthe presence of MMP-7 protein in the suprabasal cellsof the clinically healthy junctional epithelium, whereasno MMP-7 expression could be seen in periodontalpocket tissues. Therefore, they suggested that thisMMP plays an important role in the normal physiologyand antibacterial defense of junctional epithelium.19

On the other hand, elevated MMP-7 levels were re-cently found in diseased peri-implant sulcular fluid.21

In the present study, the presence of GCF MMP-7

Table 4.

Correlations Between GCF MMP-7,EMMPRIN, TIMP-1, andClinical Parameters

Clinical Parameters

MMP-7

(pg/site)

EMMPRIN

(du/site)

TIMP

(pg/site)

PD (mm) 0.173 0.613* 0.821*

CAL (mm) 0.222 0.580* 0.794*

Sites with bleeding (%) 0.116 0.657* 0.262

Sites with plaque (%) 0.085 0.387* 0.468*

GCF (ml) 0.055 0.695* 0.811*

* P <0.05.

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levels in healthy subjects suggests that MMP-7 is pro-duced by intact epithelia cells eventually for the anti-bacterial defense of junctional epithelium, whichmight reflect its beneficial roles in the defense of junc-tional epithelium. The presence of MMP-7 in the GCFof gingivitis groups might suggest that this enzyme isinvolved in the early stages of host defense againstpathogen microorganisms. We found that CP andGAgP patients had similar GCF MMP-7 levels com-pared to gingivitis and healthy groups. Our findingssuggest that MMP-7 might play an important role inthe early defense of periodontal tissues. On the otherhand, similar GCF MMP-7 levels among diseasedgroups might indicate that this enzyme does not havean important role in the pathogenesis of periodontitisunlike other MMPs that were previously shown to be in-volved predominantly in the progression of periodon-tal disease.4,29,30,32,53,54,56 When the junctionalepithelium is converted to pocket epithelium, Uittoet al.19 suggested that it could not produce MMP-7and thereby loses its defensive functions. Our presentfindings showing similar GCF MMP-7 levels amonggroups studied support and further extend thissuggestion. The presence of MMP-7 in human peri-odontitis GCF was previously demonstrated byTervahartiala et al.20 They found that MMP-7 immu-noreactive cells were only slightly expressed in CPand localized aggressive periodontitis (LAgP) gingivawhen compared to healthy controls. On the otherhand, MMP-7 has been demonstrated in vitro to medi-ate destructive events by the degradation of a varietyof matrix and non-matrix proteins.15,17 Our data sug-gest that MMP-7 is associated with the innate hostdefense in periodontal tissues. In severely inflamedtissues, more inflammatory connective tissue eventstake place in connective tissue than in the diseasedepithelium.59 It is possible that it may be for this rea-son that we could not find elevated MMP-7 in GCF inpatients with CP and GAgP. In addition to destructiveevents, similar GCF MMP-7 levels between diseasedand healthy groups suggest that defensive eventsare more prominent than tissue remodeling eventsin GAgP and CP patients.

It is usually accepted that the balance between ac-tivated MMPs and TIMPs controls the extent of extra-cellular matrix remodeling, and tissue degradation iscaused by the disruption of this balance in favor ofproteinases.3 Under pathological conditions associ-ated with unbalanced MMP/TIMP activity, changesin TIMP-1 levels could be important in the regulationof the destruction of periodontal tissues by affectingthe MMP levels in periodontal tissues.23-25 TIMP levelsin periodontal tissues are important because these en-dogenous regulators of MMP activities are involved inthe extracellular control of excessive extracellularmatrix degradation. Nevertheless, it has been previ-

ously demonstrated that TIMP-1 could inhibit the ef-fects of MMP-1, -3, -8, and -9 in inflamed periodontaltissues.30 Previously, it has been demonstrated thatTIMP-1 was present in higher levels in healthy sitesand in low levels in early inflamed sites.31-33 In con-trast, others have shown that diseased tissues ex-pressed higher levels of TIMP-1 to overcome tissuedestruction.24-30 Meikle et al.23 showed that MMPsand TIMP-1 secreting cells were present at sites whereconnective tissue remodeling takes place. In the pres-ent study, elevated GCF TIMP-1 levels in GAgP and CPpatients compared to gingivitis and healthy subjectsare in agreement with earlier findings of previous stud-ies.24-30 It might be suggested that the host is produc-ing TIMP-1 as the anti-proteolytic shield to overcomeand regulate the tissue-destructive effects of MMPs ingingival tissues.

It has been well documented that the activity of sev-eral MMPs including collagenases, gelatinases, andstromelysin increased and are activated during theprogression of periodontal disease.4,27,29,30,32,60-62

EMMPRIN was previously shown to promote MMP ex-pressionbyfibroblastsandfromtumorcells.37-40 Itcanbe involved in a wide range of physiological and path-ological processes because of its effect on variousMMP production such as MMP-1, -2, and -3.34,37 Inthe present study, we found elevated GCF EMMPRINlevels in thediseasedgroupscompared tohealthysub-jects. In other words, GCF EMMPRIN levels were foundto increase together with the severity of periodontaldisease. Therefore, by stimulating MMP productionin inflamed periodontal tissues, it is highly likely thatEMMPRIN could lead to the upregulation of the prote-olytic activity released by the recruited inflammatoryand resident cells, thereby participating in the pro-gression of periodontal disease. Immunoblotting ofdiseased GCF revealed large increases in multiplesoluble 55- to 200-kDa EMMPRIN species; the biolog-ically active form of EMMPRIN is the 55-kDa glycosy-lated form,whereas the30-kDa form isnon-glysylatedand inactive.37 The 100- to 200-kDa bands are pre-sumably dimers and trimers of 55-kDa native EMM-PRIN linked together by dysulphide bridges.41,63 Thesoluble 55-kDa functional EMMPRIN could especiallybe detected in the periodontitis and GAgP GCF. In linewith the present findings, Kasinrerk et al.64 showedthat EMMPRIN on the activated lymphocytes andmonocytes could contribute to elevated MMP levelsin inflammatory tissue destructive disease, i.e., arthri-tis.64 We suggest that elevated soluble GCF EMMPRINlevels might influence and reflect MMP action in in-flamed periodontal tissues to promote tissue degrada-tion leading to the progression of periodontal disease.

As a result, we found elevated GCF TIMP-1 andEMMPRIN levels in GAgP and CP patients comparedto the healthy group. GCF TIMP-1 and soluble

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EMMPRIN levels of both GAgP and CP groups werealso significantly higher than those of the gingivitisgroup. Moreover, GCF TIMP-1 and EMMPRIN levelswere positively correlated with the severity of peri-odontal disease. On the other hand, diseased groupshad comparable GCF MMP-7 levels to the healthygroup. The lack of elevated GCF MMP-7 levels in dis-eased groups might provide evidence that this MMP ispreferentially released into the GCF for early defen-sive purposes. Future studies investigating EMMPRINlevels and expression patterns in periodontal tissueand GCF are required to elucidate the role of this novelmolecule in the pathogenesis, course, and diagnosisof periodontal disease.

ACKNOWLEDGMENTS

This work was supported by grants from the Academyof Finland and the Helsinki University Central Hos-pital (HUCH) grants (TI020 Y0002, TYH 5316, andTYH 6104).

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Correspondence: Dr. Gulnur Emingil, Department ofPeriodontology, School of Dentistry, Ege University,Bornova-35100, _IIzmir, Turkey. Fax: 90-232-3880325;e-mail: gemingil@yahoo.com.

Accepted for publication July 19, 2006.

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