|Year : 2013 | Volume
| Issue : 3 | Page : 191-195
Estimation of interleukin-17 levels in gingival crevicular fluid from healthy individuals and patients with chronic periodontitis
Ravindra Reddy Nagireddy1, Vinay Chavan1, Madhu Babu Dandu Subramanyam2, Venkat Subba Chowdavaram Reddy1, Mohan Kumar Pasupuleti3, Kishore Kumar Avula1
1 Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, India
2 Department of Periodontics, Government Dental College, RIMS, Kadapa, India
3 Department of Periodontics, Saint Joseph Dental College, Eluru, Andhra Pradesh, India
|Date of Web Publication||29-Aug-2013|
Ravindra Reddy Nagireddy
Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: The objective of the present study was to estimate the levels of interleukin (IL)-17, the proinflammatory cytokine produced by the activated T helper 17 cells in gingival crevicular fluid (GCF) and determine its role in progression of periodontal disease.
Materials and Methods: Clinical parameters including gingival index, Russell's periodontal index and probing pocket depth were recorded for 30 subjects divided into two groups. Group 1 (healthy) comprising 10 subjects with clinically healthy periodontium and with no evidence of disease, group 2 (with periodontitis) comprising 20 subjects whose periodontium had clinical signs of inflammation, probing pocket depth ≥5 mm and with radiographic evidence of bone loss. IL-17 levels were estimated in GCF using enzyme-linked immunosorbent assay.
Results: The mean concentration of IL-17 was significantly higher in group 2 when compared with group 1. Gingival index and Russell's scores were significantly higher in group 2 when compared with group 1.
Conclusion: IL-17 was present in GCF samples from individuals in both healthy and chronic periodontitis groups and its concentration correlates positively with the extent of periodontal destruction, Russell's periodontal index scores and thus levels of IL17 in GCF may act as novel biomarker for estimating periodontal disease progression.
Keywords: Enzyme-linked immunosorbent assay, gingival crevicular fluid, interleukin-17, periodontitis
|How to cite this article:|
Nagireddy RR, Chavan V, Subramanyam MB, Reddy VS, Pasupuleti MK, Avula KK. Estimation of interleukin-17 levels in gingival crevicular fluid from healthy individuals and patients with chronic periodontitis. J NTR Univ Health Sci 2013;2:191-5
|How to cite this URL:|
Nagireddy RR, Chavan V, Subramanyam MB, Reddy VS, Pasupuleti MK, Avula KK. Estimation of interleukin-17 levels in gingival crevicular fluid from healthy individuals and patients with chronic periodontitis. J NTR Univ Health Sci [serial online] 2013 [cited 2019 Dec 8];2:191-5. Available from: http://www.jdrntruhs.org/text.asp?2013/2/3/191/117188
| Introduction|| |
Periodontitis is a disease of periodontium characterized by irreversible loss of connective tissue attachment and supporting alveolar bone. It is an inflammatory process involving innate and adaptive immune responses.  These changes often lead to an esthetically and functionally compromised dentition. The inflammatory process occurring in periodontal disease is characterized by the infiltration of leukocytes, which limits bacterial invasion. There are number of factors that promote leukocyte recruitment including bacterial products, cytokines, cross-talk between innate and adaptive immune responses, chemokines, lipid mediators and complement. The immune system is a remarkably adaptive defense system that has evolved in vertebrates to protect from invading pathogenic microorganisms. It is able to generate an enormous variety of cells and molecules capable of specifically recognizing and eliminating an apparently limitless variety of foreign invaders.
The development of an effective immune response involves lymphoid cells, inflammatory cells and hematopoietic cells. The complex interactions among these cells are mediated by a group of proteins that are collectively designated as cytokines to denote their rolls in cell-to-cell communication. Although periodontal diseases are initiated by bacteria that colonize the tooth surface and gingival sulcus, the host response is believed to play an essential role in the breakdown of connective tissue and alveolar bone, which are the key features of the disease process. An intermediate mechanism that lies between bacterial stimulation of host immune system and tissue destruction is the production of cytokines, which stimulates inflammatory events that activate effector mechanisms. These cytokines can be characterized as chemokines, innate immune cytokines and acquired immune cytokines. Although they were historically identified as leukocyte products, many are also produced by a number of cell types including keratinocytes, resident mesenchymal cells (such as fibroblasts and osteoblasts) or their precursors, dendritic cells and endothelial cells. Chemokines are chemotactic cytokines that play an important role in leukocyte recruitment and may directly or indirectly modulate osteoclast formation.  Evidence that cytokines play a critical role in host response was studied in a nonhuman primate model, which showed that inhibition of interleukin-1 (IL-1) and tumor necrosis factor (TNF) reduced the progression of periodontal bone loss and loss of attachment, , possibly due to the recruitment of inflammatory cells (notably monocytes and lymphocytes) toward the bone. 
IL-17, the proinflammatory cytokine produced by the activated T helper (Th) 17 cells,  has been implicated in numerous autoimmune and inflammatory conditions including rheumatoid arthritis, multiple sclerosis, psoriasis and inflammatory bowel disease.  Besides IL-17, which is an important regulator of host defence through neutrophil trafficking,  Th17 cells are characterized by the production of a distinct profile of effector cytokines, including IL-6, and have probably evolved to enhance host clearance range of pathogens distinct from those targeted by Th1 and Th2 responses. 
IL-17 is produced in periodontal lesions  and this cytokine is also capable of inducing the receptor activator of nuclear factor κB ligand (RANKL), the main stimulatory factor for the differentiation and activation of osteoclasts.  Vernal et al.,  showed that IL-17 was present at higher levels in the gingival crevicular fluid (GCF) and in supernatants from cellular cultures of gingival tissue from patients with periodontitis than in those from healthy subjects, suggesting a role for IL-17 in the pathogenesis of chronic periodontitis. It was demonstrated that Porphyromonas gingivalis outer membrane protein induced a significant increase in the production of IL-17 in patients with periodontitis and after that stimulation IL-17 was detected more frequently in patients with periodontitis than in those with gingivitis. ,
In the light of the above facts, this study has made an attempt to evaluate the relationship of IL-17 and periodontal disease with particular reference to the involvement of IL-17 in the pathogenesis of periodontal disease and the estimation of IL-17 levels in GCF from the patients with chronic periodontitis and healthy controls.
Aims and objectives
- To estimate the levels of IL-17 in GCF of healthy and periodontitis affected individuals.
- To determine the role of IL-17 in human periodontal disease progression.
- To explore the possibility of using IL-17 as a "novel biomarker" for periodontal disease progression.
| Materials and Methods|| |
A total of 30 patients in the age group of 20 - 50 years, who presented to the outpatient wing of periodontics department, were selected. Informed consent was obtained from the subjects and the protocol was approved by the ethics committee of our institution. Patients who had not received any periodontal treatment in the previous 6 months were included, and patients suffering from any systemic diseases, or who had received anti-inflammatory drugs, antibiotics, and neovascularization inhibitors in the previous 6 months were excluded from the study. Patients were selected randomly and categorized into two groups: Group 1 (healthy) comprising 10 subjects with clinically healthy periodontium and with no evidence of disease, group 2 (with periodontitis) comprising 20 subjects whose periodontium had clinical signs of inflammation, probing pocket depth (PPD) ≥5 mm and with radiographic evidence of bone loss.
Clinical evaluation of subjects
For each patient, gingival index (Loe and Silness, 1963), Russell's periodontal index and PPD values were recorded. All clinical measurements were carried out by a single operator. Test site for GCF sample collection was selected based on the highest scored sites in the oral cavity. GCF collection was done in the next appointment to avoid contamination of the sample.
Gingival crevicular fluid sampling method
Each selected test site for GCF collection was air dried and isolated with cotton rolls. Without touching the marginal gingiva, supragingival plaque was removed with curettes (Hu-Friedy) to avoid contamination and blocking of the microcapillary pipette. GCF was collected by placing 1 - 5 μl calibrated volumetric microcapillary pipettes obtained from Sigma-Aldrich Chemical Company, USA (Catalog No. p0549). By placing the tip of the pipette extracrevicularly (unstimulated) for 5 - 20 minutes, a standardized volume of 3 μl GCF was collected using the calibration on the micropipette from each test site. The test sites that did not express standard volume (3 μl) of GCF and the micropipettes contaminated with blood and saliva were discarded. The GCF collected was immediately transferred to aliquots and stored at –70°C till the time of the assay.
IL-17 was estimated by using Quantikine; Human IL-17 Immunoassay kit obtained from R&D systems, USA (Catalog No. D1700). The samples were analyzed using enzyme-linked immunosorbent assay (ELISA) method.
All reagents and samples were brought to room temperature before use. Assay diluents RD1-36 (100 μl) was added to each well. Standard, control, or sample (100 μl) was added per well and reagent addition was uninterrupted and completed within 15 minutes and then incubated for 3 hours at room temperature. Each well was aspirated and washed with wash buffer (400 μl), repeating the process twice for a total of three washes. After the last wash, any remaining wash buffer was removed by aspirating or decanting. IL-17 conjugate (200 μl) was added to each well, covered with a new adhesive strip and incubated for 1 hour at room temperature. Wash step was repeated as stated above and 200 μl of substrate solution was added to each well and incubated for 30 minutes at room temperature. Enzyme-substrate reaction produced a blue color and then 50 μl of stop solution was added, which produced yellow color. Using a microtiter plate reader the plate was read at the wavelength of 450 nm, which is appropriate for the substrate solution used.
Descriptive statistical analyses
Following the biochemical analysis of GCF samples, the results were obtained for all the groups and were subjected to statistical analysis. Student's t-test was used to find a significant difference between the two means. Spearman's correlation test was used to test the direction and strength of the relationship between the two variables. For pair-wise comparisons Scheffe's test was used.
| Results|| |
In the present study, when mean for different parameters was compared between the groups [Table 1], gingival index score was significantly higher in group 2 when compared with group 1 (t value 20.84, P = 0.000). Russell's index score was significantly higher in group 2 when compared with group 1 [t value 29.74, P = 0.000]. The pocket depth was significantly higher in group 2 when compared with group 1 (t value 19.74, P = 0.000). The mean concentration of IL-17 was significantly higher in group 2, i.e., 54.75 pg/ml with SD ± 12.35 when compared with group 1, i.e., 36.8 pg/ml with SD ± 10.50 (t value 3.914, P = 0.001) [Table 2].
When intercorrelation of variables in both the groups were compared, negative correlation was found between IL-17 concentration and gingival index (correlation coefficient [r] = 0.087; P > 0.716, statistically not significant), positive correlation was found between IL-17 concentration and Russell's periodontal index (r = –0.516; P < 0.02, statistically significant), between IL-17 concentration and PPD (r = 0.685; P < 0.001, statistically significant) [Table 3].
In the present study, the mean concentration of IL-17 from the sites with PPDs of 5, 6 and 7 mm was 47.42 pg/ml with SD ± 10.7, 58.46 pg/ml with SD ± 5.71, 76.53 pg/ml with SD ± 5.14, respectively [Table 4].
|Table 4: Comparison of Concentration of Il-17 in Gingival Crevicular Fluid with Different Probing Pocket Depths|
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When multiple comparisons of GCF concentrations of IL-17 were compared between PPDs of 5 and 6 mm, 5 and 7 mm, 6 and 7 mm, the results were not statistically significant between 5 and 6 mm (mean difference of –11.30, P > 0.052), 6 and 7 mm (mean difference of –18.07, P > 0.057), and statistically significant between 5 and 7 mm (mean difference of –29.11, P < 0.002) [Table 5].
| Discussion|| |
IL-17, produced mainly by cells with a Th0/Th1 profile is a proinflammatory cytokine that exhibits pleiotropic biological activities on various types of cells such as fibroblasts, endothelial cells and epithelial cells to produce other inflammatory cytokines and chemokines.  IL-17 may affect osteoclastic bone resorption by stimulating osteoblasts to produce factors that affect the activity and/or formation of osteoclasts; osteoblasts are IL-17-responsive cells and express mRNA encoding the IL-17 receptor. Hence, it is hypothesized that T cells in periodontal tissues produce IL-17 and exacerbate inflammatory periodontal disease, activating gingival fibroblasts to produce inflammatory mediators and can play a role in bone cell metabolism via T-cell-derived cytokines. Results from several clinical studies indicate that IL-17 may be involved in many inflammatory diseases. 
In the present study, the mean concentrations of IL-17 in GCF were found to increase progressively from healthy (36.87 pg/ml) to periodontitis sites (54.75 pg/ml). This result was in accordance with the results of Gonzalez et al.,  who also reported raised IL-17 concentration in GCF in periodontal diseased sites.
However, contrary to our finding, Vernal et al.,  reported a higher concentration of IL -17 in GCF of healthy samples than in periodontitis sites. According to them, volume of GCF produced in sites with probing ≥5 mm depth is higher than that in sites from healthy subjects. In the present study, GCF collection was done using microcapillary pipettes, whereas earlier studies used filter paper strips and Periotron 8000 and 6000, which can result in nonspecific attachment of the analyte to filter paper fibers ensuing in a false reduction in the detectable IL-17 levels, which underestimates the correlation of IL-17 levels to disease progression. 
In the present study, GCF IL-17 concentration when correlated with the clinical parameters, showed a positive correlation (r = –0.516, P < 0.02), (r = 0.685, P < 0.001), i.e., when clinical parameter scores increase, concentrations of IL-17 also increase. In the present analysis, the levels of IL17 increased with increase in PPD, there was a high correlation between concentration of IL-17 and PPD (r = 0.685, P < 0.001), i.e., when the pocket depth increases concentrations of IL-17 also increase. There was significant statistical difference between 5 mm and 7 mm pocket depths with mean difference of –29.11, P < 0.002. Thus, change in concentration of IL-17 remains a good indicator of periodontal disease progression. These results are in accordance with those of Gonzαlez et al. 
As per the results of Gonzαlez et al.,  it was found that the concentrations of IL-17 were higher in active sites than in ihnactive sites (P = 0.0005). Thus, IL-17 could be a good marker for periodontal disease progression.
The following conclusions were drawn from the study:
- IL-17 is present in GCF samples from healthy ndividuals and patients with chronic periodontitis.
- With increases in amount of destruction, there is substantial increase in clinical parameter and IL-17 concentration in GCF, which is directly related to pocket depth.
- IL-17 concentration correlated positively with the extent of periodontal destruction, Russell's periodontal index score; from this observation it can be postulated that IL-17 is actively involved in the progression of periodontal disease. Thus, it can be considered as novel biomarker of periodontal disease progression.
- The present study shows that IL-17 is a novel biomarker for periodontal disease progression.
In conclusion, within the limits of our study, it can be postulated that with increasing extent of periodontal destruction, increase in the levels of IL-17 in GCF was seen. However, additional longitudinal studies are needed to evaluate the concentration of IL-17 in the GCF, and periodontal diseased tissues will be beneficial in clarifying its role in the pathogenesis of periodontitis and to validate IL-17 as a "novel biomarker" of periodontal disease progression.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]