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Table of Contents   
ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 2  |  Page : 188-192
Evaluation and comparison of interleukin-8 (IL-8) level in gingival crevicular fluid in health and severity of periodontal disease: A clinico-biochemical study


1 Department of Periodontics, Rural Dental College, Loni, Rahata, Ahmednagar, Maharashtra, India
2 Department of Periodontics. M. A. Rangoonwala Dental College and Research Centre, Pune, Maharashtra, India
3 Department of Prosthodontics, Rural Dental College, Loni, Rahata, Ahmednagar, Maharashtra, India

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Date of Submission13-Jul-2011
Date of Decision06-Dec-2012
Date of Acceptance25-Dec-2012
Date of Web Publication20-Aug-2013
 

   Abstract 

Background: Cytokines play an important role in the pathology associated with chronic inflammatory diseases. Because of pro-inflammatory and neutrophil chemotactic properties, the cytokines like interleukins (IL) may play a significant role in the pathogenesis of periodontitis. The biological effects of IL-8 are relevant in this regard.
Aim: This study was done to compare the level of this molecule in gingival crevicular fluid (GCF) from patients with adult periodontitis (experimental group) and from individuals with clinically healthy gingival (control group).
Materials and Methods: GCF was collected from patients with adult periodontitis and clinically healthy gingival for 30 s using a Periopaper strip and the volume of the sample determined. Following elution of the fluid, assays for IL-8 were carried out by enzyme-linked immunosorbent assay (ELISA). The concentration of IL-8 was calculated in the original volume of GCF on each strip.
Results: The level of IL-8 in the experimental group was significantly higher than in the control group ( P < 0.01). The clinical parameters were positively correlated to IL-8, suggesting that the GCF IL-8 exhibited dynamic changes upon severity of periodontal disease ( P < 0.05).
Conclusion: These data suggest that level of IL-8 is associated with periodontal status. The level of IL-8 in GCF is valuable in detecting the inflammation of periodontal tissue.

Keywords: Cytokines, gingival crevicular fluid, interleukin-8, periodontitis

How to cite this article:
Lagdive SS, Marawar PP, Byakod G, Lagdive SB. Evaluation and comparison of interleukin-8 (IL-8) level in gingival crevicular fluid in health and severity of periodontal disease: A clinico-biochemical study. Indian J Dent Res 2013;24:188-92

How to cite this URL:
Lagdive SS, Marawar PP, Byakod G, Lagdive SB. Evaluation and comparison of interleukin-8 (IL-8) level in gingival crevicular fluid in health and severity of periodontal disease: A clinico-biochemical study. Indian J Dent Res [serial online] 2013 [cited 2020 Feb 20];24:188-92. Available from: http://www.ijdr.in/text.asp?2013/24/2/188/116675
Periodontal disease, an inflammatory disease, is also considered as an immunological disease since localized chronic inflammation associated with gingiva exhibits unique immunologic features which include elevated cellular and humoral immune responses. [1] It results from an interaction of the host defense mechanism with microorganisms in biofilm. Neutrophils are the most predominant cell population that migrates into inflammatory lesions. Besides their protective role, they are involved in tissue damage under pathological conditions because of their high contents in neutral and acid proteases and their ability to generate superoxide and other reactive oxygen derivatives.

Cytokines are small proteins that are released by various cells in the body, usually in response to an activating stimulus and induced responses through binding to specific receptors. The local host response to chronic inflammatory periodontal disease (CIPD) can be studied by biochemical analysis of gingival crevicular fluid (GCF). Recently, there has been increased interest in the role played by the interleukins (IL) in the pathogenesis of CIPD. IL-8 is a member of the IL-8 supergene family that includes other small chemotactic peptides with structural homology. It may also play a role in the pathogenesis of inflammation due to its role in recruitment and functional activation of polymorphonuclear leukocytes. [2]

The ability of IL-8 to stimulate human polymorphonucleocytes (PMN) to release myeloperoxidase, α-mannosidase, arachidonate-5-lipoxygenase, elastase, and β-glucoronidase, as well as to induce the formation of superoxide and hydrogen peroxide (H 2 O 2 ) as part of the respiratory burst emphasizes its role as a proinflammatory mediator. [2] However, continuous and excessive IL-8-mediated chemotactic and activation effects on neutrophils in the inflamed gingiva may contribute to local tissue destruction of the periodontal tissue.

Given the properties of IL-8 and its presence in the mouth via GCF, its effect on oral health, especially periodontal health, and in patients with adult periodontitis warrants further investigation. Because of its pro-inflammatory and neutrophil chemotactic properties, IL-8 may play a significant role in the pathogenesis of periodontitis.

Based on these findings, it seems reasonable to speculate that IL-8 may influence the initiation and progression of periodontitis. Hence, this study was undertaken to correlate the levels of IL-8 with the clinical parameters of chronic periodontitis.


   Materials and Methods Top


Patient selection

The present study was carried out in Department of Periodontics, Rural Dental College, Loni, in co-ordination with Department of Biochemistry, Biotechnology and Microbiology, Rural Medical College, Loni. The subjects for this study were selected from the Out-Patient Department of Periodontics, Rural Dental College and Hospital, Loni. The study conducted was a case-control study. After obtaining an informed consent, a total of 80 subjects were divided into experimental (mild 20, moderate 20, severe 20) and control (20) groups. All were free of systemic disease and were not taking medications such as antibiotics, anti-inflammatory agents, immunosuppressants, or contraceptives that could affect their periodontal status for at least 6 months prior to the study. Radiographic examination and clinical periodontal assessments included Gingival Index (GI; Loe and Silness 1963), periodontal destruction by Periodontal Disease Index (PDI; Ramfjord 1959), and clinical attachment loss. Based on the results, the subjects were divided into experimental (mild, moderate and severe) and control (clinically healthy) groups.

Collection of GCF

Based on the understanding that gravity could affect the maxillary and mandibular teeth differently, GCF was obtained only from maxillary sites. To ensure accessibility and isolation, GCF samples were obtained only from the buccal sites. [3] To avoid interference with the GCF volume, all participants were instructed not to eat anything or brush their teeth for ≥1 h before GCF sampling. For standardization, all GCF samples were obtained between 9:00 a.m. and 10:00 a.m. Subjects were seated comfortably in the dental chair with adequate lighting condition. All clinical measurements were taken 1 day prior to sampling to avoid contamination. GCF was collected according to the method defined by Rudin et al. [3] Test sites were chosen from maxillary arch to avoid contamination with saliva. After isolating the tooth with a cotton roll, supragingival plaque was gently removed with curettes, avoiding the marginal gingiva. The crevicular site was then gently dried with an air syringe prior to collection of GCF. [4] Before the sampling, an electronic gingival fluid measuring device, Periotron 8000, was switched on and allowed to warm up at room temperature. A blank Periopaper strip was placed between the counterparts of the device, setting the reading dial to zero. [5] To eliminate the potential risk of evaporation, the Periotron 8000 ® was placed in a chairside manner. The strip was placed intracrevicularly until the minimum resistance was obtained at the mesio-facial, disto-facial, or mid-facial surface of the tooth. [6] The strip was subjected to remain in place for a period of 30 s, in order to avoid cytokine secretion induced by the mechanical irritation provoked by a longer collection period. The entire amount was collected from one site only. Paper strip was immediately transferred to the Periotron 8000 ® for electronic volume quantification, i.e., within 0-2 s. A digital readout was obtained within 16 s and was converted to microliters using specialized software After each measurement, the electrodes of the device were dried using sterile cotton to eliminate the risk of any contamination between multiple readings and adjusted to zero.

Correlation between Periotron meter reading and severity of inflammation: [7]

  1. 0-40 - Healthy tissue with little or no inflammation
  2. 41-80 - Mild periodontitis
  3. 80-120 - Moderate periodontitis
  4. 120-200 - Severe periodontitis
After GCF volume measurements, strips were placed in Eppendorf tubes with 100 μl of phosphate-buffered saline with 0.05% Tween-20 (PBS-T) solution with constant agitation to elute the GCF sample. After removal of the strip, the eluted sample was centrifuged at 8000 rpm for 10 min at 4°C and the procedure was repeated three times. [2] After that, Eppendorf tubes containing the supernatants were stored at −80°C in deep freezer in Department of Biotechnology until the analysis. Paper strips that contaminated with blood or pus were discarded.

Cytokine analysis

Ten microliters of the aliquots of each GCF sample was assayed for the level of IL-8 using sandwich ELISA kit $ according to manufacturer's recommendations. IL-8 concentration (pg/μl) was calculated from the volume of GCF estimated from the calibration unit reading, according to the formula: IL-8 concentration (pg/μl) = total IL-8 (pg)/volume (μl).

Statistical analysis

The clinical parameters and the concentrations of IL-8 in control and experimental groups were calculated as subject Mean ± standard deviation. The clinical measures and the concentrations of IL-8 were compared between control and experimental groups using unpaired "t "- test. The P values reported in the "Results" section were derived from t-tests of log-transformed data. The correlations among the level of IL-8 and various clinical parameters were assessed using the Karl Pearson's correlation coefficient.


   Results Top


Subject data

[Table 1] shows the age and sex wise distribution of subjects in all the four groups under study. All the four groups consisted of 20 subjects each. Control group contained more males, compared to other groups. Moderate and severe periodontitis group had more subjects above 40 years of age.
Table 1: Age and sex wise distribution of the patients under study

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[Table 2] shows the distribution of values of clinical parameters: GI, PDI, clinical attachment level (CAL), and GCF IL-8 level. Comparison of mean values of clinical parameters and GCF IL-8 level between control and mild, moderate, and severe periodontitis groups are shown in the table. Highly significant differences were found between the statistical mean values of the experimental group and the control group (P < 0.01). The mean IL-8 level of the experimental group was significantly higher than the control group. The IL-8 level in the sites of the experimental group had GI, PDI, and CAL comparable to those of sites of the control group.
Table 2: Comparison of mean values of clinical parameters and GCF IL-8 level between control and experimental groups

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[Table 3] shows the correlation of GCF IL-8 level with GI, PDI, and CAL in the control and experimental groups. The correlation between the levels of IL-8 and clinical parameters in the control group was not significant (P > 0.05). Highly significant correlation was found between GCF IL-8 level and GI, PDI, and CAL in the mild, moderate, and severe periodontitis groups (P < 0.01).
Table 3: Karl Pearson's correlation coefficient (r) between GCF IL-8 and clinical parameters in control and experimental groups

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   Discussion Top


Inflammation occurs as a first line of defense following a bacterial challenge. Periodontal disease results from the interaction of the host mechanisms with the plaque microorganisms. There is abundant evidence that major tissue destruction in established periodontitis lesions results from recruitment of host cells via activation of monocytes/macrophages, lymphocytes, fibroblasts, and other cell types. Considerable effort has been made to study the cytokines released by different host cells when exposed to components of periodontopathic bacteria. Certain cytokines have been proposed as potentially useful diagnostic or prognostic markers of periodontal destruction. [8]

IL-8 is a potent chemoattractant for neutrophils and is thought to enhance host defense mechanism. Therefore, the levels of IL-8 were evaluated and compared in healthy and adult periodontitis subjects.

It was found in this study that higher concentration of IL-8 in GCF from adult periodontitis was detected as compared to that in periodontally healthy individuals. Several researchers have reported increased IL-8 level in GCF in periodontitis patients. In this study, GCF IL-8 level in all the three disease groups, mild, moderate, and severe, was 501.5 ± 68.53, 850.55 ± 97.29, and 1716.95 ± 305.79, respectively, while it was 210.4 ± 80.12 in the control group. On comparing all four groups, GCF IL-8 level increased significantly from control to severe periodontitis group. The results in this study are similar to the results of Giannopoulou et al. [8] who reported that IL-1β, IL-6, and IL-8 were significantly higher in the gingivitis, adult periodontitis, and early-onset periodontitis groups, as compared to healthy subjects. They also found significant correlation between GCF cytokine levels and probing depth, smoking, and stress. Their data indicated that elevated total amount of IL-1β, IL-6, and IL-8 was associated with sites showing periodontal destruction. Also, according to Mathur et al., [9] both the concentrations and total amounts of GCF IL-8 and interferon (IFN)-α were significantly correlated in diseased sites of patients with adult periodontitis, as compared to healthy sites of control patients.

In this study, on comparison between control and mild periodontitis group, GCF IL-8 level increased significantly (P < 0.01, "t " value = 12.35) and the relationship was highly significant.

On comparison between control and moderate periodontitis group, GCF IL-8 level increased significantly and this relationship was highly significant (P < 0.01, "t " value = 22.72); the same results were obtained when control and severe periodontitis group were compared. Again there was significant increase in GCF IL-8 level (P < 0.01, "t " value = 29.43).

We also compared GCF IL-8 levels among mild, moderate, and severe periodontitis groups, and the results were highly significant. On comparing mild to moderate, mild to severe, and moderate to severe periodontitis groups, it was found that GCF IL-8 level increased significantly (P < 0.01). All the results were highly significant; this shows that as the periodontal disease progresses, there is increase in GCF IL-8 level.

Data from this study indicated that the GCF IL-8 levels varied according to the severity of the periodontal disease; when the GI, PDI, and CAL scores were higher, GCF IL-8 level was high. This finding suggests that IL-8 may possess the ability to fight against infection and inflammation by attracting the neutrophils at the site of infection. This finding is similar to the results of the study by Chi-Cheng Tsai et al. [10] which show that GCF mean IL-8 level of the experimental group (75.96 ± 50.36 pg/site) was significantly higher than the control group (10.8 ± 8.11 pg/site). They also observed that the total amount of GCF IL-8 significantly decreased after therapy in adult periodontitis patients. The large amounts of inflammatory cells in the connective tissues of gingival crevices can led to release IL-8, by stimulation with bacterial products and interaction with host cells. Neutrophils form a defense curtain between the dental plaque and junctional epithelium. These neutrophils may provide protection for the periodontium. IL-8 is a chemoattractant and activator of neutrophils. It can be released during the process of phagocytosis to attract more neutrophils into the site of bacterial infection.

Within the control group, when GCF IL-8 concentration was correlated with the clinical parameters like GI, PDI, and CAL, no correlation with any of these parameters could be established. As all the subjects in the control group were healthy, they showed either no signs of inflammation or very minimal changes in the clinical parameters. Thus, better the periodontal status, the lower the concentration of IL-8.

Within the diseased group, when GCF IL-8 level was correlated with the clinical parameters, there was highly significant positive correlation with GI, PDI, and CAL. This correlation was statistically significant as P < 0.05. This indicates that if the values of GI, PDI, and CAL increase, there is significant increase in the GCF IL-8 concentration. Thus, in individuals having severe type of periodontal disease, IL-8 level was high; as the degree of periodontal disease increased, the GCF IL-8 level increased, indicating that IL-8 is highly related to the inflammatory condition of periodontium. Kurdowska et al. [11] reported that the presence of increased IL-8 concentrations within gingival tissue was correlated with sulcular depth.

Payne et al. [12] stated that because of its pro-inflammatory and neutrophil chemotactic properties, IL-8 may play a significant role in periodontal pathogenesis. They demonstrated that increased IL-8 levels in gingival tissue could result in an increased accumulation of neutrophils, which in animals has been associated with progressive periodontitis.

Concerning the various biological effects of IL-8, it is speculated that IL-8 may have both inflammation-retarding effects in the stages of gingival inflammation and inflammation-enhancing effects in the stages of periodontal destruction. Under the latter condition, disturbance of IL-8-driven neutrophil recruitment to the periodontal pocket may not only impair the potential of the host to confront the microbiological challenge, but also result in neutrophil-associated tissue destruction due to increased release of lysosomal enzymes and matrix metalloproteinases. IL-8 might thus act on migrating neutrophils in a dose-dependent mode, modulating neutrophil motility at low concentrations and contributing to the activation of neutrophil degranulation and respiratory burst at high concentrations. [13] IL-8 is particularly important in facilitating the transmigration of neutrophils and their accumulation at the surface of the subgingival plaque biofilm. Neutrophils thus play a key role as the primary effector cells to detect periodontopathogens. IL-8 plays an important role in the maintenance of local host-parasite equilibrium and in the limitation of neutrophil-associated tissue damage. Normally, IL-8 expression relates well to the pattern of neutrophil infiltration and appropriate release of IL-8 contributes to eliminating the infecting bacteria by neutrophils. Conversely, an uncontrolled, excessive release of IL-8 contributes to eliminating the excessive release of IL-8 and the resultant hyperactivity of neutrophils may result in tissue destruction. IL-8 attracts neutrophils in a manner analogous to C5a and leukotriene B4 (LTB4), but elicits activation pathways in neutrophils of matrix metalloproteinase-8, a potent collagenase for degrading host connective tissues at the sites of inflammation.

With the above background, it can be speculated that there is a positive association between the GCF IL-8 and periodontal disease severity. Thus, the level of IL-8 in GCF is valuable in detecting the inflammation of periodontal disease.

To conclude, it could be stated from this study that there seems to be a strong correlation between periodontal destruction and GCF IL-8 level. Furthermore, GCF IL-8 level increases in subjects with severe periodontal disease. More research is being directed to explore this possibility.


   Acknowledgments Top


The authors are grateful to Dr. Anand Shaker, Associate Professor, Department of Biochemistry, for his valuable help and guidance; Dr. R. Gaiker, Lecturer, Department of Biotechnology, for the timely help; and Mr. Hemant Pawar, Associate professor, Department of Medical Informatics, for helping in the statistical analysis of this study.

 
   References Top

1.Fujihashi K, Kono Y, Beagley KW, Yamamoto M, McGhee JR, Mestecky J, et al. Cytokines and periodontal disease: Immunopathological role of interleukins for B cell responses in chronic inflamed gingival tissues. J Periodontol 1993;64:400-6.  Back to cited text no. 1
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2.Chung RM, Grbic JT, Lamster IB. IL-8 and β-Glucoronidase in gingival crevicular fluid. J Clin Periodontol 1997;24:146-52.  Back to cited text no. 2
    
3.Hatipoglu H, Yamalik N, Berberoglu A, Eratalay K. Impact of the distinct sampling area on volumetric features of gingival crevicular fluid. J Periodontol 2007;78:705-12.  Back to cited text no. 3
    
4.Gamonal J, Acevedo A, Bascones A, Jorge O, Silva A. Levels of IL-1β, -8, -10 and RANTES in GCF and cell populations in adult periodontitis patients and the effect of periodontal treatment. J Periodontol 2000;71:1535-45.  Back to cited text no. 4
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5.Chapple IL, Cross IA, Glenwright HD, Matthews JB. Calibration and reliability of the Periotron 6000 for individual gingival crevicular fluid samples. J Periodontal Res 1995;30:73-9.  Back to cited text no. 5
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6.Cimasoni G. Crevicular fluid updated. Monogr Oral Sci 1983:12:III-VII, 1-152.  Back to cited text no. 6
    
7.Ciantar M, Caruana DJ. Periotron 8000: Calibration characteristics and reliability. J Periodontal Res 1998;33:259-64.   Back to cited text no. 7
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8.Giannopoulou C, Kamma JJ, Mombelli A. Effect of inflammation, smoking and stress on GCF cytokine level. J Clin Periodontol 2003;30:145-53.  Back to cited text no. 8
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9.Mathur A, Michalowicz B, Castillo M, Aeppli D. Il-1α, IL-8 and interferon- α levels in gingival crevicular fluid. J Periodontal Res 1996;31:489-95.  Back to cited text no. 9
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10.Tsai CC, Ho YP, Chen CC. Levels of IL-1β and IL-8 in GCF in adult periodontitis. J Periodontol 1995;66:852-9.  Back to cited text no. 10
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11.Kurdowska AK, Noble JM, Adcock JE. IL-8 and anti-IL-8 autoantibodies in GCF from patients with periodontitis. J Periodontal Res 2003;38:73-8.  Back to cited text no. 11
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12.Payne JB, Reinhardt RA, Masada MP, DulBois LM, Allison AC. GCF IL-8 correlation with local IL-1βlevels and patients estrogen status. J Periodontal Res 1993;28:451-3.  Back to cited text no. 12
    
13.Jin L, Soder B, Corbet EF. Interleukin-8 and granulocyte elastase in gingival crevicular fluid in relation to periodontopathogens in untreated adult periodontitis. J Periodontol 2000;71:929-39.  Back to cited text no. 13
    

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Correspondence Address:
Sushma S Lagdive
Department of Periodontics, Rural Dental College, Loni, Rahata, Ahmednagar, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.116675

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    Tables

  [Table 1], [Table 2], [Table 3]

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