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   Table of Contents    
ORIGINAL ARTICLE
Year : 2019  |  Volume : 23  |  Issue : 1  |  Page : 21-24  

Interleukin-21 and its association with chronic periodontitis


1 Department of Biochemistry, Shri B. M. Patil Medical College, Vijayapura, Karnataka, India
2 Department of Microbiology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India

Date of Submission19-Jun-2018
Date of Acceptance20-Aug-2018
Date of Web Publication3-Jan-2019

Correspondence Address:
Ms. Rani Vilas Lokhande
Department of Biochemistry, Maratha Mandal's NGH Institute of Dental, Sciences and Research Centre, Belagavi, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jisp.jisp_410_18

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   Abstract 


Context: Interleukin-21 (IL-21) is a pleiotropic cytokine, well documented to contribute to the development of Th17 cells which have been shown to play an important role in the pathogenesis of periodontitis. Periodontal disease is a chronic infection of tooth-supporting tissue. Aim: This study evaluates the saliva and serum levels of IL-21 in patients with chronic periodontitis and periodontally healthy individuals. Settings and Design: The present study was carried out in the Department of Microbiology in association with Department of Oral Medicine and Radiology, Maratha Mandal's N.G.H Institute of Dental Sciences and Research Centre, Belgavi, Karnataka. Materials and Methods: Fifty samples of each group were included in the present study. The levels of IL-21 were assessed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit and the results were expressed as pg/mL. Statistical Analysis Used: Statistical analysis was performed using SPSS 17.0 software. Data were expressed as mean ± standard deviation and interquartile ranges and comparison of controls and cases by Mann–Whitney test. Results: Serum and salivary levels of IL-21 were significantly higher in chronic periodontitis group than in controls (P < 0.001). Clinical periodontal parameters correlated positively with serum IL-21 levels. Conclusions: IL-21 is highly expressed in patients with chronic periodontitis and correlated well with clinical parameters of periodontal destruction. Therefore, IL-21 appears to play a role in tissue destruction and can be used as diagnostic biomarker in chronic periodontitis. Saliva can be considered to be a useful alternative to serum as a diagnostic sample.

Keywords: Chronic periodontitis, diagnostic biomarker, enzyme-linked immunosorbent assay, interleukin-21


How to cite this article:
Lokhande RV, Ambekar JG, Bhat KG, Dongre NN. Interleukin-21 and its association with chronic periodontitis. J Indian Soc Periodontol 2019;23:21-4

How to cite this URL:
Lokhande RV, Ambekar JG, Bhat KG, Dongre NN. Interleukin-21 and its association with chronic periodontitis. J Indian Soc Periodontol [serial online] 2019 [cited 2019 Jun 25];23:21-4. Available from: http://www.jisponline.com/text.asp?2019/23/1/21/248228




   Introduction Top


Periodontitis is an inflammatory disorder of periodontium, the supporting tissue of teeth which is characterized by the formation of pockets and loss of attachment between the tooth and gums.[1],[2] Chronic periodontitis is the common form of periodontitis that mainly occurs in young adults and is a complex disease having multifactorial etiology.[3]

Even though different microbes are known to initiate the disease, it has been proved that exaggerated and disproportionate immune response of host is responsible for damage of periodontium in affected patients. The adaptive immune response is brought about mainly due to the involvement of various pro-inflammatory cytokines produced by activated T-cells in diseased gingival and periodontal tissue.[4]

The role of several cytokines as interleukin-1 (IL), IL-6, IL-8, IL-10, and IL-12 has been evaluated in chronic periodontitis and their involvement in the destruction of periodontal tissues.[5] In recent years, interest is focused on the study of a newly identified cytokine called as IL-21.[6] Investigations have shown that activated T-cells produce IL-21 which has the ability to act on multiple cells of the immune system causing impacts on immunity to infection.[5],[7] There are not many studies conducted to find out the role of IL-21 in chronic periodontitis.

However, most of them have used gingival crevicular fluid and periodontal tissue as the clinical sample. The current study was intended to estimate IL-21 in the salivary and serum of chronic periodontitis patients and compare with those of healthy individuals and determine its diagnostic utility aid in periodontal disease.


   Materials and Methods Top


The present study conducted in the Department of Microbiology in association with the Department of Oral Medicine and Radiology in our Institute. Ethical clearance was taken from RGHUS, the Institutional Review Board of Institution before initializing study.

The study involved a total of 100 participants that included 50 cases of chronic periodontitis and 50 healthy individuals between the age ranges of 26 and 60 years belonging to both sexes. Participants with the known systemic disease, malignancies, and blood disorders, those with the habit of smoking, alcoholism, and drug abuse were excluded from the study.

In addition, those with <20 teeth and/or had undergone any dental treatment or received antibiotic therapy during the last 3 months were not considered for the study. Inclusion basis for case selection was at least 4 sites with >5 mm pocket probing depth (PD) and attachment loss. The corresponding criteria for the healthy control group were no sites with >3 mm pocket PD and attachment loss.

Written consents form was obtained from each participant before enrolling for the study. The standard operating procedure was followed for sample collection.

Blood collection

Blood samples collected in serum separator vacutainers. The site of venepuncture was cleaned with an alcohol swab. Gently, a 5 cc syringe with a 23G needle inserted in the vein and approximately 3 ml of blood collected in tubes, inverted tubes for 2–3 times to mix with clot activator in tubes. Once blood had drawn, the tourniquet was removed and kept sterile cotton swab over needle insertion and the needle was removed. Blood samples allowed to clot for 30 min at room temperature. Tubes transported to the laboratory and centrifuged at 15,000 rpm for 10 min. Serum samples were collected in multiple aliquots and stored at - 80°C until laboratory analysis.

Saliva collection

Navazesh spitting method was followed for collection of unstimulated saliva.[8]

The saliva collection procedure was explained to the participants. Participants asked to abstain from food or any drinks for minimum 1 h. Participants asked to rinse the mouth with water. Sterile wide-mouth containers were labeled and given for saliva collection. Participants were asked to accumulate the saliva in the floor of the mouth and asked to spit in a container for every 60 s. In this way, 2–3 mL saliva collected and transported to the laboratory on an ice pack. The saliva samples were vortexed for 2 min. The saliva samples transferred in 10 mL Falcon tubes and centrifuged for 10 min at 10,000 rpm. The supernatants were collected and stored as multiple alliquotes at - 80°C.

Laboratory measures

IL-21 levels were assessed by means of a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Krishgen Biosystems 15375 Ashley Ct. Whittier, CA Lot No 1211015). Manufacturer's instructions were strictly adhered while performing the assay.

Briefly, 100 μL of standards and samples were added to labeled microtiter wells and incubated for 2 h at room temperature. Unbound antigen removed by washing step. Following this, the wells were kept for 2 h with 100 μL detection antibody at room temperature. Moreover, washing step repeated as mentioned earlier. Then, to each well, 100 μL Avidin-HRP was added and waited for 30 min.

Wells have washed again and then incubated with tetramethylbenzidine substrate for 20 min. Then, 100 μL of stop solution was added to stop the reaction and optical density was read at 450 nm in microplate LISA reader. The IL-21 concentration was determined as pg/ml. Results of the test samples were calculated using the standard curve produced in an assay.


   Results Top


This study includes 100 participants which included 50 periodontally healthy controls and 50 cases with chronic periodontitis [Table 1].
Table 1: Age and sex distribution pattern of chronic periodontitis patients and healthy individuals

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The IL-21 levels quantified using ELISA technique from samples of each participant from control and study groups. The values of serum IL-21 in control groups ranged from 15.8 to 193.3 pg/mL (mean 65.34 pg/mL). Salivary levels of IL-21 in controls varied from 15.0 to 86.0 pg/mL (mean 21.98 pg/mL). Serum levels of IL-21 in chronic periodontitis patient ranged from 67.0 to 694.5 pg/mL (mean 497.78 pg/mL). Salivary IL-21 levels were 32.0–812.0 pg/mL (mean 214.02 pg/mL) in chronic periodontitis [Table 2].
Table 2: Comparison between interleukin-21 levels of healthy and chronic periodontitis groups by Independent samples t-test

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The mean IL-21 levels were compared in chronic periodontitis patients and periodontally healthy participants by Independent sample t-test. Statistical analysis was significant with mean levels across test groups with P < 0.005. Clinical parameters for chronic periodontitis PD and clinical attachment loss (CAL) were assessed for correlation with IL-21 levels in the serum and saliva. It was found that there was significant correlation of PD with serum IL-21 levels (rs= -0.341, P =0.016) but no significance could attached to IL-21 levels in both test fluids with CAL [Table 3].
Table 3: Pearson's correlation to compare pocket probing depth, loss of attachment with the Interleukin-21 levels in serum and in saliva

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In [Table 4], the present study was compared with other previous studies.
Table 4: The comparison of results from various studies

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   Discussion Top


Among the inflammatory diseases, chronic periodontitis is commonly found in human beings. The disproportionate adaptive response toward microbial pathogens present in the dental plaque is known to be the reason for the destruction of periodontal tissues in diseased patients.[9] Both innate and adaptive immune responses are involved in the progression of periodontal disease.

Literature survey shows the participation of various T-cells subsets in periodontal diseases. There are three subtypes of T helper cells – Th1, Th2, and Th17 cells. Investigations have shown that T-cells produce inflammatory cytokines which are causative agents of the symptoms and complications of periodontitis.[10] Recently, the role of Th17 cells and IL-21 has been the topic of investigation in periodontitis and several other systemic infectious disorders.[11],[12] IL-21 was discovered in 2000 as a CD4 + T-cell-derived cytokine. It belongs to common gamma chain of cytokines family.

It is a pleiotropic cytokine and has the tendency to impact on immunity. IL-21 involved in the development of Th17 cells, which play a major role in the disease process of periodontitis.[13],[14],[15] We investigated the utility of serum and salivary IL-21 levels in patients with chronic periodontitis and periodontally healthy individuals which has not been tried so far.

Most of the previous studies used the gingival tissue, gingival crevicular fluid for detection of IL-21 levels using different methods including immunohistochemistry, Western blot, polymerase chain reaction, and ELISA.[16] The most of these studies showed significantly raised levels of IL-21 in chronic periodontitis patients when compared to healthy individuals.

Several investigators have shown IL-21 levels correlated significantly with periodontal parameters.[5],[13] The literature survey has shown only one study, wherein levels of salivary IL-21 was decreased after induced physical stress.[17] In [Table 4], the present study was compared with other previous studies.

The present study analyzed IL-21 levels in serum and salivary samples by ELISA technique which is the most sensitive, speedy, easy to carry out, and reproducible method.[18] The study found a significant difference between periodontally healthy and chronic periodontitis patients for both serum and salivary IL-21 levels (P < 0.001). The levels of serum IL-21 in chronic periodontitis cases were correlated negatively with probing pocket depth by Pearson's correlation.

it was interesting to find that serum and salivary IL-21 levels showed no correlation with CAL. Saliva is a diagnostic medium having many advantages in comparing with other body fluids. The proteins and RNA contents of both saliva and serum are similar.

Collection of saliva sample is convenient and safe for both technicians and patients. Saliva is readily available, noninvasive, and easier to handle.[19],[20],[21],[22] There are upcoming newly methods are arising for diagnosis of periodontitis. At present, detection of the periodontal pathology depends predominantly on the clinical and radiological examinations, which are useful in tracing data of the past disease, present periodontal health, but suggest incomplete information about diseases and sites at threat for the future periodontal disease risk.

Several diagnostic parameters in the saliva have proposed and use as an analytical aid for periodontal pathology. Early diagnosis and management reduce the severity and possible complication of the disease process.


   Conclusions Top


These data concluded that IL-21 levels were significantly increased in chronic periodontitis patients and linked with clinical periodontal parameters of tissue destruction.

Therefore, IL-21 levels might responsible for tissue damage processes that characterize chronic periodontitis. IL-21 levels used as one of the diagnostic and prognostic aids in chronic periodontitis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci 1993;694:72-7.  Back to cited text no. 8
    
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Nizam N, Basoglu OK, Tasbakan MS, Nalbantsoy A, Buduneli N. Salivary cytokines and the association between obstructive sleep apnea syndrome and periodontal disease. J Periodontol 2014;85:e251-8.  Back to cited text no. 13
    
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Yi JS, Cox MA, Zajac AJ. Interleukin-21: A multifunctional regulator of immunity to infections. Microbes Infect 2010;12:1111-9.  Back to cited text no. 14
    
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Kratholm SK, Iversen MB, Reinert L, Jensen SK, Hokland M, Andersen T. Interleukin-21 receptor signalling is important for innate immune protection against HSV-2 infections. PLoS One 2013;8:e81790.  Back to cited text no. 15
    
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Mootha A, Malaiappan S, Jayakumar ND, Varghese SS, Toby Thomas J. The effect of periodontitis on expression of interleukin-21: A systematic review. Int J Inflam 2016;2016:3507503.  Back to cited text no. 16
    
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Moreira A, Bacurau RF, Napimoga MH, Arruda AF, Freitas CG, Drago G, et al. Salivary IL-21 and IgA responses to a competitive match in elite basketball players. Biol Sport 2013;30:243-7.  Back to cited text no. 17
    
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Yolken RH. Enzyme-linked immunosorbent assay (ELISA): A practical tool for rapid diagnosis of viruses and other infectious agents. Yale J Biol Med 1980;53:85-92.  Back to cited text no. 18
    
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Patil PB, Patil BR. Saliva: A diagnostic biomarker of periodontal diseases. J Indian Soc Periodontol 2011;15:310-7.  Back to cited text no. 19
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Isaza-Guzmán DM, Cardona-Vélez N, Gaviria-Correa DE, Martínez-Pabón MC, Castaño-Granada MC, Tobón-Arroyave SI, et al. Association study between salivary levels of interferon (IFN)-gamma, interleukin (IL)-17, IL-21, and IL-22 with chronic periodontitis. Arch Oral Biol 2015;60:91-9.  Back to cited text no. 22
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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