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Amplified Crevicular Leukocyte Activity in Aggressive Periodontal Disease

¹ Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, Boston, MA, USA;
² Institute of Physiological Chemistry, University of Marburg, Germany; and
³ Department of Periodontology, School of Dental Medicine, University of Münster, Germany;

Correspondence: ^* corresponding author, Bussardstrasse 6, D-59071 Hamm, Germany, rainer_buchmann{at}yahoo.de

	ABSTRACT

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Exaggerated neutrophil responses are a critical component in the pathogenesis of periodontal disease. We investigated whether leukocyte activity in aggressive periodontitis (AP) is increased compared with that in chronic periodontitis (CP) by gingival crevicular fluid (GCF) analysis of myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH), cathepsin D (CD), and elastase-alpha-1-proteinase inhibitor complex (alpha-1-EPI) before and 6 months after therapy. Initial AP neutrophil responses were significantly amplified compared with those in CP (MPO, 3.2-fold; beta-NAH, 37.5-fold; CD, 2.2-fold; alpha-1-EPI, 1.4-fold; p < 0.05). Surgical therapy resulted in a significant reduction of GCF markers compared with non-surgical treatment. However, the changes in clinical parameters were not different between AP and CP (P > 0.05). Analysis of the results suggests that the local inflammatory response in AP is characterized by increased release of inflammatory mediators of neutrophil origin into the GCF. Analysis of the data further suggests that surgical therapy is a more predictable method for removal of the pro-inflammatory etiology.

Key Words: aggressive periodontitis • inflammation • gingival crevicular fluid • PMN enzymes • protease-complexed elastase • host defense mechanisms

	INTRODUCTION

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Periodontal inflammation may affect and worsen systemic conditions such as cardiovascular disease, pre-term labor, diabetes mellitus, and other clinical syndromes associated with leukocyte-mediated pathology. Several prominent neutrophil-specific molecules have been implicated as amplifiers of the inflammatory response (Battino et al., 1999; Yamalik et al., 2000). The myeloperoxidase-hydrogen peroxide-chloride system of the neutrophil, as part of innate host defense, possesses potent antimicrobial activity (Ihalin et al., 1998). The liberation of the 150-kDa-protein myeloperoxidase, a potent oxidative enzyme from the azurophilic granules of neutrophils, in combination with reactive oxygen species, such as O₂^–, gives rise to hypochlorous acid (HOCl). In homeostasis, inflammation, and during phagocytosis, glycoside sulphatases and acid hydrolases secreted by neutrophils (Shikhman et al., 2000) may be released from terminal lysosomes serving as storage bodies (Tjelle et al., 1996). The lysosomal acid hydrolase, beta-N-acetyl-hexosaminidase, is released into gingival crevicular fluid during neutrophil-mediated inflammatory events in the gingiva, subsequent to phagocytosis by the neutrophil. Salivary beta-NAH-glycosidase activity correlates with the degree of inflammation, and follows resolution of inflammation after treatment (Nieminen et al., 1993). The 52-kDa lysosomal aspartate-endopeptidase, cathepsin D, is released from the primary azurophilic granules of neutrophils into the extracellular compartment. Cathepsin D is functional in a wide variety of tissues during remodeling or regression, and in apoptosis. Cathepsin D is found in fibroblasts and macrophages of the subepithelial connective tissue, and in desquamating junctional epithelial cells of the gingival crevice (Ayasaka et al., 1993). These molecules in GCF have been used as an endpoint marker of tissue degradation and cell lysis (Lamster, 1991; Hasilik, 1992). In rheumatoid arthritis (Momohara et al., 1997) and acute exacerbation of respiratory tract infections (Groeneveld et al., 1997), the elastase-alpha-1-proteinase inhibitor complex, a 53-kDa-serine proteinase, has been shown to be an early inflammatory marker.

Recently, aggressive periodontitis was redefined as a complex disease exhibiting microbial alteration and cellular dysfunction that differentiate the underlying molecular mechanisms of its pathogenesis from chronic periodontal disease (Armitage, 1999). The release of granule components from infiltrating leukocytes, such as lysosomal enzymes and reactive oxygen species, which are normally intended to degrade ingested microbes, can also lead to tissue degradation and amplification of the inflammatory response, with continued recruitment of new leukocytes. In localized aggressive periodontitis in particular, it has been demonstrated that uncontrolled neutrophil recruitment and activation can lead to the aberrant release of an array of noxious agents intended to fight the bacteria, with the potential for causing further tissue damage (Pouliot et al., 2000). In aggressive periodontitis, most sites respond following therapy, but certain sites fail to resolve, and actually continue to experience active disease (American Academy of Periodontology, 2000), suggesting incomplete removal of etiology.

With this background, the null hypothesis tested was that leukocyte activity in periodontal tissues of aggressive periodontal disease is not significantly different from that in chronic periodontitis.

	MATERIALS & METHODS

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Patients
Twenty-five patients with aggressive periodontitis (AP) (Armitage, 1999) and nine subjects with chronic periodontitis (CP) were recruited from the Department of Periodontology, University of Münster. AP patients were younger (AP, 39.7 ± 8.6 yrs vs. CP, 49.2 ± 7.1 yrs) and exhibited more severe disease radiographically, with evidence of intrabony defects exceeding 50% (AP) and 30% (CP), respectively, of the root length. Another inclusion criterion was probing depths > 5 mm in at least 8 sites within each quadrant. AP subjects displayed generalized severe periodontal tissue destruction, loss of periodontal support inconsistent with age, persistent gingival inflammation, increasing probing depths, and progressing tooth mobility (American Academy of Periodontology, 2000). Exclusion criteria included pregnancy, periodontal therapy or antibiotics in the previous six months, any systemic condition that might have affected the progression or treatment of periodontitis, and the need for antibiotic pre-medication before dental therapy. A written informed consent was obtained from each volunteer. The study protocol was approved by the Institutional Review Board of the Medical Faculty of the University of Münster.

Biochemical Analyses
Myeloperoxidase
GCF sampling, analysis, and recovery and validation protocols have been described previously (Buchmann et al., 2002). A 10-µL quantity of the diluted GCF sample was added to 0.1 M citric acid buffer, pH 5.5, containing 0.125% Triton-X-100 solution and 0.1 mM hydrogen peroxide. The oxidation of H₂O₂ was performed with 0.8 mM -Dianisidin as substrate. After 7 min of incubation at 21°C, the samples turned brown, and the reaction was stopped by the addition of glycine/NaOH, pH 10.4. The myeloperoxidase activity of the GCF samples was determined spectrophotometrically at a wavelength (wv) of 405 nm (reference wv 650 nm) on uncoated 96-well microtiter plates with the use of a microplate reader. The total MPO activity was calculated in duplicate and expressed in µU/GCF sample.

Beta-N-acetyl-hexosaminidase
A 10-µL quantity of the diluted GCF sample was eluted in 25 µL of 0.9% NaCl and 0.1 M citric acid buffer, pH 4.6, and a 25-µL quantity of p-nitrophenyl-N-acetyl-beta-D-glusoaminide (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) was added. The reaction was allowed to proceed at 37°C for 30 min and then was stopped with 225 µL of 0.4 M Glycine/NaOH stopping buffer, pH 10.4. The concentration of p-nitrophenolate was assessed at a wv of 405 nm (reference wv 492 nm). The total beta-NAH activity was calculated in duplicate and expressed in µU/GCF sample (Hasilik, 1992).

Cathepsin D
Cathepsin D was determined with [¹⁴C] hemoglobin used as the substrate. The diluted sample (10 µL) was incubated in a total volume of 200 µL in 0.1 M Na-acetate buffer, pH 3.65, with 10 µg [¹⁴C] hemoglobin (specific activity = 240 pg/cpm). After 6 hrs of incubation at 37°C, the reaction was stopped by the addition of 0.5 mL 1% (w/v) casein solution and 0.5 mL 25% (w/v) trichloroacetic acid. The samples were centrifuged for 5 min at 14,000 x g, and the radioactivity of the supernatant was quantified in a liquid scintillation counter. The total cathepsin D activity was determined in duplicate, and average values were calculated in ng.

Elastase-alpha-1-proteinase inhibitor complex
The total amount of complexed, functionally inactive, PMN elastase was determined with the Merck-Immunoassay (Merck KgaA, Darmstadt, Germany). In 2 x 5 µL of the diluted samples, the alpha-1-EPI-complex was bound to 20 µL of the solid-phase fixed anti-PMN-elastase antibody. After 10 min of incubation at 37°C, a 20-µL quantity of the alpha-1-proteinase-antibody conjugate was added. The alpha-1-EPI complex was determined at a wv of 492 nm by the addition of 50 µL 4-p-nitrophenylphosphate as substrate, and 150 µL of 2 N NaOH as stopping reagent. The total amount of elastase-alpha-1-proteinase inhibitor complex was computed from 492-nm absorbance readings with the use of a calibration curve of 4 different standards and expressed in ng.

Periodontal Intervention
All patients were monitored at baseline and 6 mos after periodontal treatment. The clinical examination included measurement of probing depth at 6 sites per tooth (PD), clinical attachment level at 6 sites per tooth (CAL), the gingival index (GI) and the plaque index (PI), bleeding on probing (BOP), and gingival crevicular fluid volume (GCFv) quantified by means of a Periotron 8000. The periodontal treatment protocols have been previously reported (Buchmann et al., 2002). In AP patients, 3 x 500 mg amoxicillin (Ratiopharm GmbH, Blaubeuren, Germany) and 3 x 250 mg metronidazole (Artesan GmbH, Lüchpow, Germany) were administered systemically as an adjunct to therapy, while in CP subjects, no antibiotics were prescribed.

Statistical Analysis
Data analysis and statistical tests were performed on the patient level. A site-level analysis was applied to differentiate surgical from non-surgical treatment outcomes. Significant changes of the GCF parameters and clinical parameters after therapy were analyzed by the Wilcoxon Signed-rank test. Differences between AP and CP were analyzed by the Mann-Whitney U-test. The GCF parameters were computed as the total amount collected onto the paper strips in 30 sec and plotted for both visits as medians, with the Q1-Q3 quartiles, the minima and the maxima. Statistical significance was defined as P < 0.05.

	RESULTS

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Periodontal Parameters
Both of the patient population groups exhibited a significant treatment effect, as evidenced by changes in PD, CAL, GI, PI, BOP, and GCFv (P = 0.08 to 0.0001, inter-group comparisons, Table 1). AP patients demonstrated more severe disease, in general, as evidenced by deeper pockets and more attachment loss. However, at 6 mos, there were no differences between groups for any parameter except GI (P = 0.0001). The significant change of GCF volumes in both treatment groups (AP, P = 0.0001; CP, P = 0.008) indicated a marked decrement of inflammatory exude after therapy.

View larger version (19K): [in this window] [in a new window]   Figure. Initial GCF neutrophil responses in AP were significantly amplified compared with those in CP. (A) Myeloperoxidase activity (MPO); (B) beta-N-acetyl-hexosaminidase activity (Beta-NAH); (C) cathepsin D (CD); and (D) elastase-alpha-1-proteinase inhibitor complex (Alpha-1-EPI). PRE = outset. 6 M = 6 mos post-therapy. The post-treatment levels remained significantly different between AP and CP (not CD and Alpha-1-EPI).

Cathepsin D
Baseline CD was 2.2-fold higher in AP than in CP (3.1 in AP vs. 1.4 ng in CP). Periodontal treatment significantly lowered CD in both AP and CP, but AP CD levels remained elevated compared with those in CP (P = 0.004) (Table 2).

Elastase-alpha-1-proteinase inhibitor complex
Baseline alpha-1-EPI was 1.4-fold higher in AP (94.0 in AP, and 68.2 ng in CP). The decrease of alpha-1-EPI after therapy was significant in both AP (54 ng) and CP (21.2 ng). The reduction of GCF markers was significantly different between AP and CP (P = 0.002) (Table 2).

Non-surgical vs. Surgical Therapy
One hundred thirty-two periodontal sites were treated surgically in the AP group and 40 sites in the CP group, while 68 sites were treated by scaling and root planing in the AP group and 32 sites in the CP group. At sites with PD > 6 mm treated by surgery, a significantly greater reduction of leukocyte markers was observed for both AP and CP compared with scaling and root planing alone. In AP, the reduction of MPO (P = 0.0001), CD (P = 0.002), and alpha-1-EPI (P = 0.001) significantly differed between the surgical and non-surgical groups, whereas no differences were noted for beta-NAH (P = 0.027) and GCFv (P = 0.426). In CP, the changes were more pronounced (Table 3).

	DISCUSSION

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Hypochlorous acid (HOCl), the product of an MPO-catalyzed reaction, inactivates alpha-1-proteinase inhibitor by oxidizing a methionine residue that is essential for its biological activity (Travis et al., 1994). Due to the inactivation of the antiproteinase shield by HOCl, MPO provides appropriate conditions for the action of latent proteases (Yamalik et al., 2000). Our findings support the concept that severe periodontal inflammation, as seen in AP, is due in part to an abnormally high PMN response. Since the inflammatory exudates (GCF volume) were similar in untreated AP and CP, our findings suggest that the oxygen-dependent metabolism in neutrophils may serve as a qualitative marker for the differentiation of periodontal inflammation.

The profile of the lysosomal glycosidase beta-NAH that participates in the matrix degradation process is characterized by an up-regulation (37.5-fold) in AP compared with CP. These differences cannot be explained by altered signal transduction events governing the PMN responses in AP. Beta-NAH is considered to be an outer-membrane-associated lipoprotein of P. gingivalis (Lovatt and Roberts, 1994). Unwanted contributions of microbial-origin beta-NAH from the periodontal environment may accelerate neutrophil-generated tissue damage. Resolution of inflammation and clinical healing induced a concomitant down-regulation of beta-NAH significant for both disease groups. The striking differences for beta-NAH between AP and CP after therapy, where the microbial impact on inflammation is less pronounced, support the hypothesis of a pivotal role of neutrophil action in severe inflammation.

CD is linked to PMN secretion at the endpoint of inflammation (Lamster, 1991), as the lesion begins to resolve. We found a 2.2-fold increase of CD in AP compared with CP. The release of CD during acute inflammation simultaneously inactivates endogenous proteinase inhibitors, allowing uncomplexed, functionally active CD to contribute to ongoing tissue damage (Tervahartiala et al., 1996). CD concentrations decrease with resolution of inflammation. The differences between AP and CP disappeared after completion of periodontal therapy.

The functional stability of the alpha-1-EPI complex depends on oxidation by free radicals and the action of proteolytic enzymes destroying the reactive alpha-1-PI loop. We demonstrate here significantly different alpha-1-EPI levels at sites from AP or CP patients. These observations provide evidence of potent leukocyte actions in excessive inflammation. The decrement of the alpha-1-EPI complex in AP following therapy is consistent with previous findings in CP (Flemmig et al., 1996), where exudating neutrophils were reduced by additional topical application of antimicrobials during therapy.

Probing depths were found to be associated with leukocyte activity in the gingival crevice. Regardless of the type of disease, observed changes in GCF markers at sites with PD > 6 mm treated by surgery were more pronounced compared with non-surgically-treated sites. The relationship of alpha-1-EPI to PD has recently been confirmed (Meyer et al., 1997). In contrast, the location of sample sites in the oral cavity does not affect leukocyte activity and the resulting GCF parameters (data not shown). The need for surgical intervention in the treatment of AP is controversial. It remains unclear why surgical access would yield better results in the resolution of inflammatory markers, but it is likely related to better access for the removal of etiologic factors in the treatment of the disease.

In conclusion, analysis of the data presented here supports the concept that effective treatment of periodontal disease can be measured with the use of biochemical markers of PMN activity. Our results stress the apparent tight regulation of PMN activity during periodontal inflammation, wound healing, and resolution, until homeostasis is achieved.

	ACKNOWLEDGMENTS

 
The project was supported both by Merck KgaA, Darmstadt, and by institutional funds from the University of Münster, Germany. The authors appreciate the inspiration and support of C.N. Serhan, Harvard Medical School, Boston, in the preparation of this manuscript. We are indebted to M. Karpisch-Tölke, MTA, University of Münster, for her comprehensive cooperation and assistance in performing the biochemical assays. We acknowledge the statistical assistance of Martha E. Nunn, Boston University.

Received for publication August 6, 2001. Revision received July 2, 2002. Accepted for publication July 9, 2002.

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Journal of Dental Research, Vol. 81, No. 10, 716-721 (2002)
DOI: 10.1177/154405910208101012


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