Potential Role of Vascular Endothelial Growth Factor, Interleukin-8 and Monocyte Chemoattractant Protein-1 in Periodontal Diseases

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En Lee,Yi-Hsin Yang,1 _{Ya-Ping Ho, Kun-Yen Ho, andChi-Cheng Tsai}

Graduate Institute of Dental Sciences and 1Graduate Institute of Oral Health Sciences, College of Dental Medicine, Kaohsiung Medical University,

Kaohsiung, Taiwan.

Host-mediated immunoinflammatory pathways activated by bacteria lead to destruction of the periodontal connective tissues and alveolar bone. The objective of this study was to elucidate the activation of the inflammatory processes in periodontal disease by quantitative assessment of cytokines and periodontopathogens. Gingival crevicular fluids (GCF) and subgingival plaque samples were collected from patients with chronic periodontitis and gingivitis and from periodontally healthy sites. Vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), and interleukin 8 (IL-8) in GCF were analyzed by enzyme-linked immunosorbent assay. Periodontopathogens, including Bacteroides

forsythus, Campylobacter rectus, Porphyromonas gingivalis and Prevotella intermedia, were

analyzed by immunofluorescence and dark-field microscopy. There was significantly more VEGF and IL-8 in chronic periodontitis and gingivitis sites than in periodontally healthy sites. There were significant positive correlations between the concentrations and total amounts of VEGF and IL-8 in chronic periodontitis and gingivitis sites, and between the levels of periodontopathogens and the total amounts of VEGF, MCP-1 and IL-8. These data indicate that inflammatory processes induced by periodontopathogens and the activation of certain cytokines (VEGF, MCP-1, IL-8) in periodontal diseases may be relevant to host-mediated destruction in chronic periodontitis.

Received: April 11, 2003 Accepted: June 6, 2003

Address correspondence and reprint requests to: Dr. Chi-Cheng Tsai, College of Dental Medicine, Kaohsiung Medical

University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan.

E-mail: chchts@kmu.edu.tw

Key Words: vascular endothelial growth factor, cytokines,

chemokines, periodontopathogens, periodontal disease (Kaohsiung J Med Sci 2003;19:406–15)

Periodontal disease is generally understood to be an infectious disease caused by microbial challenge which, although essential in the process, is insufficient on its own to cause disease [1]. The microbes induce the host

immunoinflammatory response, which leads to per-turbation of connective tissue and bone metabolism. The combination of environmental and genetic risk factors and a susceptible host leads to chronic perio-dontitis characterized by inflammatory destruction of connective tissue, loss of periodontal attachment, and resorption of alveolar bone. Host inflammatory mediators and pathways of tissue destruction have been extensively investigated in recent years. Bacte-rial metabolites and related molecules may also

indirectly stimulate inflammatory infiltrate: monocytes may release interleukin (IL)-1 [2], tumor necrosis factor alpha (TNF-α) [3], prostaglandin E2 (PGE2) [4], IL-8 [5],

and matrix metalloproteinases [6] to activate processes in endothelial and other periodontal cells. The aim of this study was to investigate the relationship of periodontal disease to vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), and IL-8.

VEGF, also known as vascular permeability factor [7] or vasculotropin, is a multifunctional mediator that stimulates endothelial cell proliferation, secre-tion of proteolytic enzymes, chemotaxis and migrasecre-tion, all of which are necessary for angiogenesis. Angio-genesis (the process by which new blood vessels are produced from established vessels) contri-butes to the severity of inflammation as a result of the ability of new blood vessels to transport pro-inflammatory cells to the lesion and supply nutrients and oxygen to the inflamed tissues, thus increasing the potential substrate for the production of cytokines, adhesion molecules, and other factors of inflammation [8].

MCP-1 belongs to the family of CC chemokines [9], which have two adjacent cysteine residues. CC chemokines are chemotactic for monocytes and a small subset of lymphocytes. MCP-1 is one of the most thoroughly characterized CC chemokines [10]. Its expression has been detected in a number of different disease states in vivo and has been implicated in several pathologic processes, including atherosclerosis [11,12], delayed hypersensitivity reactions [13], host response to tumors [14], arthritis [15], gingivitis [16], and osseous inflammation induced by bacterial infection [17]. Injection of MCP-1 in vivo leads to the predominant recruitment of monocytes [18]. MCP-1 expression by endothelial cells may be important in the regulation of integrin expression in leukocytes. MCP-1 produced by endothelial cells could diffuse into monocytes in the capillary lumen and activate a d h e s i o n m o l e c u l e s t h a t f a c i l i t a t e m o n o c y t e attachment. Production of MCP-1 by mononuclear phagocytes in the interstitial tissue could represent an important amplification step. Monocyte chemotactic activity in the crevicular fluids of adult periodontal disease patients increases with the severity of the disease [18], suggesting that MCP-1 may contribute to the observed increased infiltration of monocytes into periodontal tissue.

IL-8 is a chemoattractant cytokine produced by a variety of tissue and blood cells with a distinct target specificity for the neutrophil [5]. Neutrophils represent the major inflammatory infiltrate in periodontitis [19]. In diseases with neutrophil dysfunction, periodontal tissue is lost very rapidly. Connective tissue constituents are effectively degraded by neutrophil enzymes, released upon activation. IL-8 is involved in the initiation and progression of periodontal disease [20].

The purpose of this study was to elucidate the activation of inflammatory processes in periodontal disease by quantitative assessment of vascular remodeling and proliferation-related cytokines (VEGF), chemokines (MCP-1, IL-8), major periodonto-pathogens (Bacteroides forsythus, Campylobacter rectus,

Porphyromonas gingivalis, Prevotella intermedia), and

routine clinical parameters.

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Patient selection

Thirty-six patients referred to the Department of Periodontics, Chung-Ho Memorial Hospital, for the treatment of periodontitis or gingivitis or routine oral examination were selected. Some patients had a his-tory of non-surgical treatment. However, none had undergone scaling or root-planing in the past 3 months except daily tooth brushing. Patients with compli-cated medical histories, including diabetes, rheuma-toid arthritis, and cardiac valvular defects, were ex-cluded from the study. None of the subjects had taken medications such as anti-inflammatory agents, antibiotics, immunosuppressants, or contraceptives that could affect their periodontal status for at least 3 months prior to the study. Six clinically healthy dental personnel were selected for comparison. Radiographic examination and assessment of clinical parameters, including plaque index (PLI) [21], gingival index (GI) [22], probing depth (PD) [23], and probing attachment level (PAL), were performed. The selected sites were categorized into chronic periodontitis (GI > 0, PD > 3 mm, PAL ≥ 3 mm), gingivitis (GI > 0, PD ≤ 3 mm, PAL ≤ 2 mm), and periodontally healthy (GI = 0, PD ≤

3 mm, PAL ≤ 1 mm) sites.

Collection of GCF samples

In each patient, clinical measurements and gingival crevicular fluid (GCF) samples were taken from the

mesiobuccal sites of selected maxillary premolar-mo-lar teeth (excluding those with crowns or cervical restorations). After isolating the tooth with a cotton r o l l , t h e P L I a n d G I w e r e r e c o r d e d a n d t h e supragingival plaque was removed without touching the gingiva [24]. The crevicular site was then gently dried with an air syringe. GCF was collected with a Periopaper gingival fluid collection strip (IDE Interstate, Amityville, NY, USA) positioned in the pocket or sulcus for 30 seconds. The amount of sample collected was quantitated using a Periotron 6000 (Harco Electronics Limited, Winnipeg, MB, Canada). Each strip was then eluted in 500 µL elution media contain-ing physiologic saline and 0.1% Tween 20. The eluate was stored at –70°C until assayed.

Collection of subgingival plaque samples

Subgingival plaque samples were initially collected using sterile paper points (Johnson Fine Absorbent Point; Johnson and Johnson, Warminster, PA, USA) extending into the pocket or sulcus for 10 seconds, then using sterile curets [25]. The collected samples were immediately transferred into micro test tubes (Eppendorf AG, Hamburg, Germany) containing 1 mL of phosphate-buffered saline (PBS; pH 7.2) and sent to the laboratory.

Microbiologic processing of plaque samples

Antisera to B. forsythus 335, C. rectus 372, P. gingivalis 381, and P. intermedia ATCC 25611 were prepared in rabbits as described by Lai et al [26]. Subgingival plaque samples were vortexed and homogenized, and the sample suspension was smeared on glass slides, air-dried, and acetone-fixed for 10 minutes, then stored at –70°C. Fixed slides were warmed in a 40°C incuba-tor for 10 minutes before processing. Specific rabbit antisera diluted 1:320 were applied to the slides at 37°C for 30 minutes, and the slides were washed with P B S , i n c u b a t e d f o r 1 h o u r w i t h f l u o r e s c e i n isothiocyanate-labeled goat-anti-rabbit immunoglobu-lin (ICN Pharmaceuticals Inc., Aurora, OH, USA) di-luted 1:40, then washed in PBS and blotted; cover slips were added over 90% glycerol in PBS.

Slides were then examined using dark-field microscopy (Olympus PIMC-35 ADS-2, ShinJuki, Japan) to obtain total bacterial counts per high-power field. Fluorescein-labeled cells in the same field were then counted with appropriate filters to determine the proportions of B. forsythus, C. rectus, P. gingivalis,

and P. intermedia. At least three high-power fields containing a minimum of 300 cells were examined for each antiserum. The proportion of each perio-dontopathogen was the number of fluorescent bac-teria divided by the total bacbac-terial count in the same field.

Quantitation of VEGF, MCP-1 and IL-8

Mediators associated with vascular endothelial activation were analyzed using double antibody sand-wich enzyme-linked immunosorbent assay (Quan-tikine®

, R&D systems, Minneapolis, MN, USA). The concentrations of VEGF, MCP-1 and IL-8 in GCF samples were calculated by comparing sample dilu-tion curves with those of recombinant VEGF, MCP-1 and IL-8 standards. Total amounts of each cytokine were obtained by multiplying concentrations and GCF volumes [27].

Statistics

Statistical evaluation was carried out using ANOVA a n d T u k e y - K r a m e r t o c o m p a r e t h e l e v e l s o f periodontopathogens in subgingival plaque and of cytokines in GCF. Pearson’s correlation coefficients were used to show the association between cytokine variables. R-squares, usually considered as the p r o p o r t i o n o f t h e t o t a l v a r i a n c e s e x p l a i n e d by the explanatory variables, were computed for cytokines and periodontopathogens. Furthermore, a hierarchical stepwise regression strategy was applied to select the important variables for disease severity (i.e. PD and PAL). First, cytokine variables were selected based on stepwise selection procedures with a p value of less than 0.05. Next, the chosen important variables along with periodontopathogen variables were entered into another regression model for second-run selection.

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The 42 subjects contributed samples from 140 selected sites divided into three categories: chronic perio-dontitis, gingivitis, and periodontally healthy.

P. gingivalis was the predominant species in chronic

periodontitis, followed by B. forsythus, C. rectus and

P. intermedia. Summary statistics for

periodonto-pathogen levels in the subgingival plaque samples by site are illustrated in Table 1.

Table 2 shows the summary statistics for cytokine concentrations in the GCF samples by site. VEGF was detected in 74 of 96 and 26 of 44 site samples in diseased and healthy sites, respectively. VEGF con-centration in gingivitis sites was significantly higher than that in periodontally healthy sites (Tukey-Kramer). However, there were no significant differ-ences in VEGF concentration between chronic peri-odontitis sites and periodontally healthy sites or be-tween chronic periodontitis sites or gingivitis sites, although VEGF concentration was lower in chronic periodontitis than in gingivitis sites. Significant dif-ferences were found between the total amount of VEGF in chronic periodontitis sites and periodontally healthy

sites, and between gingivitis sites and periodontally healthy sites, respectively (p < 0.0001).

MCP-1 was detectable in only 37 of 96 samples from diseased sites and was not detectable in periodontally healthy sites. MCP-1 concentrations were significantly higher in chronic periodontitis sites than in gingivitis sites (p < 0.0001). There was also a significant difference in the total amounts of MCP-1 in chronic periodontitis sites and gingivitis sites.

IL-8 was detected in all 140 samples. There was no difference in IL-8 concentration in any of the three categories (ANOVA). However, the total amount of IL-8 in chronic periodontitis sites was significantly higher than in periodontally healthy sites (p < 0.0001).

Table 1. Summary statistics for periodontopathogen levels in subgingival plaque samples by site, expressed as mean percentage (standard deviation)

n B. forsythus C. rectus P. gingivalis P. intermedia

AP 47 20.32 (5.05) 16.48 (4.43) 31.49 (5.05) 13.93 (4.69)

G 49 6.22 (2.51) 4.70 (2.74) 10.66 (6.78) 4.38 (2.74)

H 44 0.73 (1.22) 0.35 (0.79) 1.39 (2.24) 0.21 (0.57)

ANOVA * * * *

Tukey-Kramer post-hoc comparison

AP vs H * * * *

G vs H * * * *

AP vs G * * * *

AP = chronic periodontitis sites; G = gingivitis sites; H = periodontally healthy sites. *_{Statistically significant at overall α = 0.05.}

Table 2. Summary statistics for cytokine levels in gingival crevicular fluids (GCF) by site, expressed as mean values (standard deviation)

VEGF MCP-1 IL-8

GCF volume Concentration Total amount Concentration Total amount Concentration Total amount

n (µL) n (ng/mL) (pg/site) n (ng/mL) (pg/site) n (ng/mL) (pg/site)

AP 47 0.87 (0.27) 28 16.38 (18.88) 13.45 (15.31) 28 8.53 (16.82) 5.97 (11.59) 47 214.73 (215.09) 178.32 (180.41)

G 49 0.60 (0.24) 46 21.86 (25.13) 11.56 (9.79) 9 0.40 (1.22) 0.19 (0.49) 49 242.20 (225.00) 128.83 (92.37)

H 44 0.42 (0.15) 26 9.33 (9.66) 3.61 (3.96) 0 0 (0) 0 (0) 44 213.11 (168.97) 77.83 (53.73)

ANOVA * * * * * *

Tukey-Kramer post-hoc comparison

AP vs H * * * * *

G vs H * * *

AP vs G * * *

VEGF = vascular endothelial growth factor; MCP-1 = monocyte chemoattractant protein-1; IL-8 = interleukin-8; AP = chronic periodontitis

Correlations among the concentrations of VEGF, MCP-1 and IL-8 with clinical parameters are shown in Table 3. VEGF concentration was not correlated with any of the clinical parameters, while the total amount of VEGF was positively correlated with PLI, GI, PD and PAL. MCP-1 concentration and total amount were only positively correlated with GI. IL-8 concentration was negatively correlated with PLI, but the total amount of IL-8 was positively correlated with GI, PD and PAL.

A significant relationship was noted between the concentrations of VEGF and IL-8, which can be ex-pressed as: IL-8 concentration = 67.2232 + 8.029924 ×

VEGF concentration (r = 0.801, p < 0.0001; Figure A). Similarly, there was a significant correlation between the total amounts of VEGF and IL-8: total amount of IL-8 = 23.1422 + 9.63561 × total amount of VEGF (r = 0.796, p < 0.0001; Figure B).

R-squared analysis showed that cytokines com-bined with periodontopathogens contributed more to PD and PAL than either cytokines or periodonto-pathogens alone (Table 4). Stepwise regression analy-sis revealed that the total amount of VEGF combined with C. rectus was the most relevant combination for PD and PAL (Table 5).

Table 3. Correlation among cytokine levels and clinical parameters (Pearson’s correlation coefficient, r)

VEGF MCP-1 IL-8

Concentration Total amount Concentration Total amount Concentration Total amount

PLI 0.096 0.291* _{0.146 0.069 –0.212}* _0.008

GI 0.188 0.401* _0.272* _0.202* _{–0.026 0.201}*

PD 0.133 0.509* _{0.135 0.153 –0.018 0.297}*

PAL 0.130 0.502* _{0.131 0.140 –0.008 0.317}*

VEGF = vascular endothelial growth factor; MCP-1 = monocyte chemoattractant protein-1; IL-8 = interleukin-8; PLI = plaque index; GI =

gingival index; PD = probing depth; PAL = probing attachment level. *_{Statistically significant (p < 0.05). All site samples were pooled for}

analysis.

Figure. Relationship between levels of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) in 100 gingival crevicular fluid samples. Each point represents a single site. (A) The regression line is IL-8 concentration = 67.2232 + 8.029924 × VEGF concentration; r = 0.801, p < 0.0001. (B) The regression line is IL-8 total amount = 23.1422 + 9.63561 × VEGF total amount;

r = 0.796, p < 0.0001.

A

B

Total amount of VEGF (pg/site)

Total amount of IL-8 (pg/site)

1000 900 800 700 600 500 400 300 200 100 0 0 20 40 60 80

D

VEGF and IL-8 were detected in a high proportion of GCF samples from chronic periodontitis patients. MCP-1 was detected only in some samples from chronic periodontitis and gingivitis sites, especially severely inflamed sites, but was not detectable in periodontally healthy sites. VEGF concentration was significantly higher in gingivitis sites than in healthy sites. However, there was no significant difference in VEGF concentra-tion between chronic periodontitis sites and periodontally healthy sites.

Investigators have reported the presence of VEGF in GCF from chronic inflammatory periodontal dis-ease [28]. They report a higher volume of GCF and total amount of VEGF in chronic periodontitis sites than in periodontally healthy sites, which agrees with

our findings. VEGF is a multifunctional cytokine asso-ciated with neovascularization and angiogenesis dur-ing inflammation and wound healdur-ing and can be de-tected in endothelial cells, neutrophils [29], plasma cells, junctional epithelial cells, pocket epithelial cells, and gingival epithelial cells. It stimulates the proli-feration of endothelial cells and the secretion of enzymes that degrade proteins, and induces chemo-taxis and migration of leukocytes, all of which are necessary for angiogenesis. PGE2 in GCF is related to

the active destruction of periodontal ligament [30]. Ben-Av et al reported that PGE2 is a potent factor in

VEGF synthesis, and VEGF synthesis induced by PGE2

and IL-1α is considered an important mechanism of inflammatory angiogenesis [31].

The significantly positive correlation between the levels of IL-8 and VEGF in this study may indicate that

Table 4. R-squared analysis for probing depth and probing attachment level

Clinical parameter GCF substance R2

Probing depth

Bacteria 0.6281

Cytokine 0.3361

Bacteria + cytokine 0.7077

Probing attachment level

Bacteria 0.5765

Cytokine 0.3148

Bacteria + cytokine 0.6588

GCF = gingival crevicular fluid. Classified into chronic periodontitis, gingivitis, and periodontally healthy categories. Bacteria represents the proportion of subgingival plaque periodontopathogens. Cytokine includes the concentration (ng/mL) and total amount (pg/site) of vascular endothelial growth factor, monocyte chemoattractant protein-1, and interleukin-8.

Table 5. Stepwise regression analysis for probing depth and probing attachment level

Variable Parameter estimate Standard error F p

Probing depth

VEGF concentration –0.0340 0.0080 24.92 0.0001

VEGF total amount 0.0885 0.0140 39.88 0.0001

C. rectus 0.1551 0.0128 147.69 0.0001

R2 _{= 0.6739}

Probing attachment level

VEGF concentration –0.0455 0.0099 20.47 0.0001

VEGF total amount 0.1000 0.0174 32.87 0.0001

C. rectus 0.1845 0.0159 134.85 0.0001

R2 _{= 0.6457}

IL-8 also takes part in inflammatory angiogenesis. Pearson’s correlation coefficient showed that the cor-relation between IL-8 and VEGF was greater in gingi-vitis sites than in chronic periodontitis sites, indicat-ing that inflammatory angiogenesis may be more ac-tive in the early stages of inflammation. The total amount of VEGF was positively correlated with all the tested periodontopathogens. This implied that periodontopathogens may induce the secretion of VEGF by interaction with neutrophils, endothelial cells and other epithelial cells within gingival tissues during microbial challenge, thus promoting inflam-mation and neovascularization. Hanatani et al re-ported that in adult periodontitis patients, the concen-tration of VEGF in GCF was higher than that in blood, indicating that VEGF is synthesized locally in the periodontal tissues [32]. Johnson et al also reported that VEGF might be the etiologic factor for gingivitis and its progression to periodontitis by initiating the expansion of the vascular network [33]. Future investigations into the expression of VEGF receptors within periodontal tissues and its relationship with different levels of periodontal disease and health are necessary to elucidate the biologic role of VEGF, whether in periodontal tissue destruction or periodontal health maintenance.

MCP-1 is chemotactic to monocytes and a small subset of lymphocytes [10]. It can be induced directly by bacteria and viruses and their metabolites, or indi-rectly by external signals such as IL-1, TNF, and other growth factors. In our study, MCP-1 was detected only in severely inflamed gingival sites (positively correlated with GI). This agreed with the findings of Yu et al [34], whose report concluded that the expression of MCP-1 was up-regulated in bacteria-associated gingivitis sites and there was nearly no expression of MCP-1 in gingival tissues with nearly no inflammation. MCP-1 is expressed by endothelial cells and mononuclear phagocytes in inflamed gingival tissues stimulated by plaque bacteria [35]. MCP-1 expressed by endothelial cells plays an important role in the regulation of leukocyte integrins [36,37]. MCP-1 produced by activated endothe-lial cells may diffuse into monocytes within the microcirculation. Activated adhesion molecules may promote the adhesion of monocytes to the vascular wall, resulting in diapedesis. Mononuclear phagocytes in the interstitial tissues may stimulate the production of MCP-1 to recruit more monocytes to promote inflammation.

Our results showed that both the concentration and total amount of B. forsythus were positively corre-lated with MCP-1. Jiang et al reported that peripheral blood mononuclear cells stimulated by P. gingivalis could produce extensive amounts of MCP-1 and IL-8 [38]. Jiang and Graves also reported that P. gingivalis lipopolysaccharides could stimulate bone-derived cells such as osteoblasts to release large amounts of MCP-1 in vitro [39]. However, the in vivo bacterial biofilm is comprised of many different bacterial communities in microenvironments of different pH, oxygen tension and electrical potential, and which expresses more resistant factors against host defense mechanisms than planktonic cultures. The implication of the positive correlation between B. forsythus and MCP-1 is that

B. forsythus might possess a certain influence on the

production of MCP-1 by host cells in vivo. The detailed mechanism should be investigated in future studies.

IL-8 is a member of the CXC chemokine family [9, 40], which has an intervening amino acid between the first two cysteines. CXC chemokines are chemotactic to neutrophils [5] and can be released from endothelial cells, gingival fibroblasts, neutrophils, monocytes, and phagocytes in the gingival crevice. Our previous study reported that GCF showed dynamic changes in the total amount of IL-8 according to the severity of perio-dontal disease [20]. In other words, the total amount of IL-8 in GCF from adult periodontitis sites was higher than that from periodontally healthy sites (but the difference in concentrations was not significant). After phase I periodontal therapy, the total amount of IL-8 was significantly reduced. We found that IL-8 could be detected in all samples and that there was no significant difference in the concentration of IL-8 in chronic periodontitis, gingivitis and periodontally healthy sites. However, the total amount of IL-8 in chronic periodontitis sites was significantly higher than that in periodontally healthy sites (Tukey-Kramer post-hoc comparison; p < 0.0001). Mathur et al found that the mean total amount of IL-8 ± standard deviation was 190 ± 130 pg/site in the diseased sites of adult periodontitis subjects, 70 ± 60 pg/site in the healthy sites of adult periodontitis subjects, and 100 ± 100 pg/site in healthy sites of periodontally healthy subjects [41]. Our results were slightly lower, with total IL-8 amounts of 178 ± 180 pg/site in chronic periodontitis sites, 129 ± 92 pg/site in gingivitis sites, and 78 ± 54 pg/site in periodontally healthy sites, but were eight times higher than the results in our

previous report [20]. We believe that this is because of the different methods used for GCF collection. Our previous study used an extrasulcular method to col-lect GCF, which would not stimulate the epithelium of the gingival crevice, and the Periopaper strip did not have direct contact with the pocket epithelium or the base of the gingival crevice. Mathur et al [41] and our present study used intrasulcular methods that ex-tended into the periodontal pocket and gingival crevice. The total amount of IL-8 was positively correlated with all tested periodontopathogens, among which

B. forsythus showed a higher correlation than P. gingivalis. Gingival epithelial cells insulted by P. gingivalis 381 may inhibit the aggregation of IL-8,

which decreases the host periodontal defense mechanism in local chemokine paralysis [42]. However, this can be overcome when the bacterial biofilm is completely established because large amounts of in-flammatory infiltrate such as neutrophils and lymphocytes aggregate to eliminate the microbial chal-lenge [43].

R-squared analysis showed that cytokine and chemokine factors combined with periodonto-pathogens contributed more to PD and PAL than either cytokine factors or periodontopathogens alone. This suggests that the host response, combined with microbial insult, has greater influence on disease severity than either host response or microbial insult alone. Stepwise regression analysis showed that the total amount of VEGF and C. rectus was the most relevant cytokine-bacteria combination for disease severity (i.e. PD and PAL).

I n c o n c l u s i o n , o u r d a t a i n d i c a t e t h a t t h e inflammatory processes induced by periodonto-pathogens and the activation of certain cytokines (VEGF, MCP-1, IL-8) may be relevant to host-mediated destruction in chronic periodontitis.

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The authors acknowledge the research grant (NSC-88-2314-B-037-093) from the National Science Council of Taiwan, the Republic of China. We would also like to thank Dr. C.H. Lai of the Microbiology Testing Laboratory, School of Dental Medicine, University of Pennsylvania, USA, for his advice and instruction on the immunofluorescence procedure for subgingival plaque samples.

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