Effectiveness of Non-Surgical Treatment to Reduce IL-18 Levels in the Gingival Crevicular Fluid of Patients with Periodontal Disease

Periodontal disease progression is due to a combination of factors including the presence of periodontopathic bacteria and high levels of proinflammatory cytokines, such as interleukin (IL)1b (1), tumor necrosis factor (TNF)-α (2) and more recently IL-18 (3). IL-18 is a proinflammatory cytokine related to the IL-1 family that is produced by Kupffer cells, activated macrophages, keratinocytes, intestinal epithelial cells, osteoblasts and adrenal cortex cells (4). The primary functions of IL-18 include the induction of IFN-g and TNF-α in T cells and natural Killer (NK) cells (5), upregulation of Th1 cytokines including IL-2, granulocyte macrophage colony stimulating factor and IFN-g (4). Effectiveness of Non-Surgical Treatment to Reduce IL-18 Levels in the Gingival Crevicular Fluid of Patients with Periodontal Disease


INTRODUCTION
Periodontal disease progression is due to a combination of factors including the presence of periodontopathic bacteria and high levels of proinflammatory cytokines, such as interleukin (IL)-1b (1), tumor necrosis factor (TNF)-α (2) and more recently IL-18 (3).
IL-18 is a proinflammatory cytokine related to the IL-1 family that is produced by Kupffer cells, activated macrophages, keratinocytes, intestinal epithelial cells, osteoblasts and adrenal cortex cells (4).The primary functions of IL-18 include the induction of IFN-g and TNF-α in T cells and natural Killer (NK) cells (5), upregulation of Th1 cytokines including IL-2, granulocyte macrophage colony stimulating factor and IFN-g (4).
It plays an important role in the innate immunity and it has been shown to induce not only T helper (Th) 1 but also Th2 cytokines, such as IL-4, IL-5, IL-10 and IL-13 (6).IL-18 is described to be upregulated in different chronic diseases, including type I diabetes, lupus and Crohn's disease (7).
Recently, our research group demonstrated that IL-18 in the gingival crevicular fluid (GCF) is higher in periodontitis patients and is associated with periodontopathogens from the red complex (11).Therefore, it is reasonable to believe that IL-18 might be involved in periodontal destruction and progression, making it interesting the analysis of the local effect of periodontal treatment.Thus, the aim of this study was to evaluate the effectiveness of the non-surgical periodontal treatment in reducing the GCF levels of IL-18 from inflamed periodontal sites.

MATERIAL AND METHODS
Fourteen subjects participated in this study.Nine patients (mean age: 48.8 SD ± 7.4 years) with generalized chronic periodontitis and 5 gingivitis controls subjects (mean age: 43.6 SD ± 11.8 years) who were attending the Dental School of the State University of Rio de Janeiro (UERJ), Rio de Janeiro, Brazil.Pedro Ernesto University Hospital's Ethics Committee (UERJ, Rio de Janeiro, Brazil) approved this study.The participants had no ongoing systemic disease or infections and gave written informed consent to participate.
GCF was collected with an intracrevicular washing device modified from Salonen and Paunio (14).The sites to be sampled were isolated with cotton rolls and dried gently with an air syringe.Supragingival plaque was carefully removed before sampling.Each pocket selected was washed 5 times with 5 mL of phosphate buffered saline (PBS) during continuous aspiration.Samples from the same type of site in each person were pooled together, diluted with PBS up to 1 mL and immediately centrifuged at 3,000 x g for 10 min.The supernatant was collected and frozen at -20ºC, until analysis.
In the patient group, samples were taken from 5 sites with pocket depth ≥5 mm and GI 1-2 (PP -periodontitis sites from periodontitis patients) and 5 sites with pocket depth ≤3 mm and GI 1-2 (GP -gingivitis sites from periodontitis patients).In the control group, 5 sites with pocket depth ≤3 mm with GI 1-2 (GG -gingivitis sites from gingivitis patients) were sampled.
IL-18 was measured in GCF using a commercially available ELISA kit (Quantikine HS; R&D Systems, Minneapolis, MN, USA and MBL, Nagoya, Japan), following the respective manufacturer's instructions.IL-18 amounts were expressed as picograms (pg).
The patients with periodontitis received nonsurgical periodontal treatment, which comprised instructions about oral hygiene and supra-and subgingival debridement (scaling and root planing) under local anaesthesia.The treatment took, on average, 4 sessions of 40 min, within 2 weeks.The control group received the same treatment except for the subgingival scaling.The scaling and root planing was performed with manual instruments (Gracey and McCall Curettes; Hirschfield Files, HU-Friedys Mfg.Co. Inc.) and by a single trained operator.Re-evaluation was performed 30 days after completion of non-surgical treatment.
The significance of the differences between day 0 and 30 AT, as well as between GP and PP sites, was calculated with the Wilcoxon signed-rank test.The significance of the differences between gingivitis and periodontitis patients was calculated with the Mann-Whitney U test.Probability values higher than 0.05 were considered as not significant.
The clinical parameters (PI, GI, PPD, AL) and the total amounts of IL-18 from each patients are presented B.O. de Campos et al.
in Table 3.

DISCUSSION
The present study revealed a significant reduction in the GCF levels in inflamed periodontal sites after nonsurgical periodontal treatment.To the best of our knowledge, there is only one study evaluating the effectiveness of the periodontal treatment in the levels of IL-18 (9).The effect of periodontal treatment has been investigated in others pro-inflammatory cytokines.Gamonal et al. (15) showed that the periodontal treatment reduced the levels of IL-1b, IL-8 in the GCF.Goutoudi et al. ( 16) also observed a reduction of the total amount of IL-1b after periodontal therapy.
IL-18 levels in GCF from inflamed periodontal disease sites have been investigated.Johnson and Serio (8) demonstrated that IL-18 concentration was higher in gingival biopsies adjacent to sites where the probing depth was >6 mm when compared to healthy sites.Pradeep et al. ( 9) showed a similar result, but in GCF, where the levels of IL-18 were higher before treatment and decreased after non-surgical treatment.When the groups were analyzed separately, the levels of IL-18 were higher in patients with gingivitis and in the deepest pockets of patients with periodontitis.We believe that this might have occurred due to the small number of patients in the study.Orozco et al. (3) found IL-18 to be increased in shallow inflamed sites in periodontitis patients when compared with gingivitis sites in control patients.Our research group obtained similar results, but we also found that the red complex was significantly increased in the PP sites when compared with the GG and GP sites, thus suggesting the existence of an association between the presence of red complex species and higher levels of IL-18 (11).
In general, non surgical therapy was effective to control the progression of periodontal disease.However, attachment loss after treatment and during maintenance may occur in the absence of signs of chronic inflammatory periodontal disease (17).It is reasonable to believe that pro-inflammatory cytokines might be involved in the subclinical activation of the periodontal inflammation.We understand that there were certain limitations to the current study including the limited number of subjects and samples evaluated, but this pilot study will encourage further investigations about the importance of the IL-18 in the disease progression.
In conclusion, this study showed that non-surgical treatment is effective in reducing GCF levels of IL-18 from inflamed periodontal sites.Table 1.Mean values (±SD) for plaque index (PI), gingival index (GI), probing pocket depth (PPD), attachment level (AL) and total amounts of IL-18 (IL-18) expressed in picograms (pg) in 5 gingivitis sites from subjects with gingivitis alone (GG), and in 9 gingivitis sites (GP) and 9 periodontitis sites (PP) from patients with chronic generalized periodontitis.

Table 3 .
Individuals values for Plaque Index (PI), Gingival index (GI), probing pocket depth (PPD), attachment level (AL) and total amounts of IL-18 (IL-18) expressed in picograms (pg) in and from 9 sites from gingivitis sites (GP) 9 periodontitis sites (PP) from patients having chronic generalized periodontitis and 5 gingivitis sites from subjects with gingivitis alone (GG).
# Significance of the difference before and after treatment; p<0.05.§ Significance of the difference between GP and PP.Σ Significance of the difference between GG and PP.