Indian Journal of Dental ResearchIndian Journal of Dental ResearchIndian Journal of Dental Research
HOME | ABOUT US | EDITORIAL BOARD | AHEAD OF PRINT | CURRENT ISSUE | ARCHIVES | INSTRUCTIONS | SUBSCRIBE | ADVERTISE | CONTACT
Indian Journal of Dental Research   Login   |  Users online: 4499

Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size         

 


 
Table of Contents   
ORIGINAL RESEARCH  
Year : 2012  |  Volume : 23  |  Issue : 3  |  Page : 348-352
Estimation of the level of tumor necrosis factor-α in gingival crevicular fluid and serum in periodontal health and disease: A biochemical study


Department of Periodontics, Priyadarshini Dental College & Hospital, Chennai, India

Click here for correspondence address and email

Date of Submission08-Mar-2011
Date of Decision04-Aug-2011
Date of Acceptance07-Nov-2011
Date of Web Publication11-Oct-2012
 

   Abstract 

Tumor Necrosis Factor-α(TNF-α), a "major inflammatory cytokine" not only plays an important role in periodontal destruction, but also is extremely toxic to the host. Till date, there are not many studies comparing the levels of TNF-α in GCF and serum and its relationship to periodontal disease.
Aim: Hence, an attempt is made to estimate the level of TNF-α in GCF and serum, its relationship to periodontal disease, and to explore the possibility of using the level of TNF-α in GCF as a biochemical "marker" of periodontal disease.
Materials and Methods: 60 subjects participated in the study and were grouped into control, gingivitis and periodonititis groups. The GCF and serum samples were assayed for TNF-α levels by Enzyme Linked Immunosorbent Assay (ELISA) method.
Results: Showed elevated levels of TNF-α in group II and III subjects as compared to healthy controls in both GCF and serum, suggesting an association between periodontal disease and levels of TNF-α.
Conclusion: It remains a possibility that the absence or low levels of TNF-α in GCF might indicate a stable lesion and elevated levels might indicate an active site but only longitudinal studies taking into account, the disease "activity" and "inactivity" could suggest the possibility of using TNF-α in GCF as an "Indicator" of periodontal disease.

Keywords: GCF, Periodontitis, TNF-α

How to cite this article:
Gokul K. Estimation of the level of tumor necrosis factor-α in gingival crevicular fluid and serum in periodontal health and disease: A biochemical study. Indian J Dent Res 2012;23:348-52

How to cite this URL:
Gokul K. Estimation of the level of tumor necrosis factor-α in gingival crevicular fluid and serum in periodontal health and disease: A biochemical study. Indian J Dent Res [serial online] 2012 [cited 2018 Aug 20];23:348-52. Available from: http://www.ijdr.in/text.asp?2012/23/3/348/102221
Though micro-organisms have been implicated as the etiological factor for periodontal disease, pathology of these inflammatory lesions have been attributed not only to bacterial products, but also to chemical mediators released by the host cells, as a result of inflammatory and immune reactions. [1] Oral micro-organisms trigger the endogenous pathways of tissue degradation by activating the host cells to produce and release inflammatory mediators and cytokines.

These inflammatory mediators and cytokines manifest potent pro-inflammatory and catabolic activity and may play a key role in local amplification of the immune response as well as in periodontal tissue breakdown. [2] Among the chemical mediators released by the host cells in response to inflammatory stimulus, the most important mediators that have a crucial role in the pathogenesis of periodontal disease are cytokines, prominent among them being interleukin -1b (IL-b), prostaglandin- E2 (PGE2) and tumor necrosis factor - a (TNF-α) .

Cytokines are soluble proteins produced by the structural and inflammatory cells, into the extracellular fluid, where they exert their effects on the same cells (autocrine activity) or on neighboring cells (paracrine activity) by interacting with specific receptors. Cytokines play an important role in numerous biological activities including proliferation, development, differentiation, homeostasis, regeneration, repair and inflammation.

However, the role of cytokines in maintenance of tissue homeostasis requires delicate balance between catabolic and anabolic activities.

TNF are grouped among the "major inflammatory cytokines", which are characteristically produced at the sites of inflammation by infiltrating mononuclear cells. Tumor necrosis factor "family" includes two structurally and functionally related proteins, TNF-α or cachectin, mainly produced by monocytes and / or macrophages and TNF-β or lymphotoxin, a product of lymphoid cells.[3]

Among the hallmarks of TNF-α is the extremely pleiotropic nature of its action, which could be ascribed to the presence of TNF receptors virtually on all cells leading to an activation of multiple signal transduction pathways, kinases and transcription factors. [3] Kull and Jacobs have reported that there are about 1000 to 3000 receptors virtually on all cells. TNF-α once produced and secreted, will bind to TNF receptor present in all plasma membrane of most of the cells throughout the body.

There are 2 types of receptors for TNF-α, i.e. TNF-α R1 and TNF-α R2. It has been reported that TNF-α R1 activation is responsible for mediating LPS toxicity and cell toxicity, and an activation of TNF-α R2 is responsible for cellular proliferation. [3]

TNF-α is pleiotropic cytokine that can improve the host defense mechanism by mediating inflammation and increasing immune cell function, at the same time, it can also induce disease through TNF-α toxicity by causing tissue injury, catabolic illness and mediating shock. Various pathological conditions are associated with the production of high levels of TNF-α like, septic shock syndrome, cachexia (eg. HIV tuberculosis, cancer), autoimmune diseases, hepatitis, leukemia, myocardial ischemia, organ transplantation rejection, multiple sclerosis, rheumatoid arthritis and meningococcal septicemia. Many people die from a complication of infectious disease called "septic shock syndrome" which is triggered by TNF-α. [4]

Thus, the systemic elevation of TNF-α level is extremely toxic to the host and hence has been termed as the "Suicide hormone". [5]

Role of TNF-α in periodontal disease

TNF-α is a proinflammatory cytokine and it induces the secretion of collagenase by fibroblasts, resorption of cartilage and bone, and has been implicated in the destruction of periodontal tissues in periodontitis. [6] TNF-α induces the synthesis of IL-1 and PGE 2, also activates osteoclasts and thus induces the bone resorption. [7] TNF-α has synergistic effects with the bone resorptive actions of IL- 1b. Studies have reported elevated levels of serum TNF-α in patients with periodontitis. [8]

It has also been reported that specific local blockage of IL-1 and TNF-α significantly reduced periodontal destruction in a monkey periodontitis model. In situ, hybridization and immunohistochemistry were used to show that TNF-α mRNA was abundant in macrophages and T-cells of the gingival tissues of patients with moderate to severe periodontitis. These findings support the hypothesis that TNF-α could have some possible role in the inflammatory process and subsequent tissue destruction in periodontal disease. Evaluation of the contents of GCF is a promising, non-invasive method for determining the tissue changes in periodontium. [9] The suitability of using cytokine TNF-α in GCF as a possible indicator of periodontal disease was first assessed by Rossomando. [10] An aim of this study is to estimate the level of TNF-α in GCF and serum, and to explore its relationship to periodontal disease.


   Materials and Methods Top


60 patients with an age range of (35 - 50 yrs) with the mean age of 40 yrs attending the outpatient section in the Department of Periodontics, were selected for the study. Patients with systemic illness and have been on medication in the past 6 months, menstruating females, females on oral contraceptives, habitual smokers were excluded from the study.

The subjects were categorized based on Ramfjord's Periodontal Disease Index and radiographic evidence of bone loss into 3 groups. Group I (control group) consisted of 20 subjects, having clinically healthy periodontium with no evidence of disease and with Ramfjord's Periodontal Disease Index as 0. Group II (gingivitis) - comprised of 20 subjects, whose gingiva showed clinical signs of inflammation but there was no evidence of attachment loss. Their Intra Oral Peri Apical radiographs did not show any bone loss, and the subjects scored between 1 and 3 with Ramfjord's Periodontal Disease Index. Group III (chronic generalized periodontitis) - 20 subjects showed clinical signs of gingival inflammation and attachment loss with radiographic evidence of bone loss, and subjects who scored between 4 and 6 with Ramfjord's Periodontal Disease Index.

Samples of gingival crevicular fluid were obtained by placing color coded, calibrated, volumetric micro capillary pipettes, extra crevicularly. The pipette had a range from 0-5 micro liter and was obtained from Sigma Chemicals Company.

2 ml of blood was collected from the antecubital fossa by venipuncture using 20 gauge needle and 2ml syringes and immediately transferred to the laboratory. Samples were allowed to clot for 1 hour at room temperature centrifuged for 10 minutes (4°C) and serum was extracted. Collected GCF and serum samples were stored at - 70°C before used for assay procedure. Serum and GCF samples were assayed for TNF-α levels by using a TNF-α reagent kit, with catalog no CK500500, obtained from Antigenix America Inc, USA.


   Results Top


Statistical analysis for the study were assessed, based on the following data: TNF-α levels in GCF and serum [Table 1], statistical significance using ANOVA for TNF-α levels in GCF and serum [Table 2] and [Table 3], Dunn's multiple comparison for pair wise comparison in TNF-α levels in GCF and serum between the 3 groups using Least Significant Difference test (LSD) at 5% level of significance [Graph 1], [Graph 2] and [Graph 3] and Spearman's correlation coefficient between the level of TNF-α, in GCF and serum [Table 4].
Table 1: Comparing the mean, and median values of TNF - α in GCF and in serum for all the 3 groups

Click here to view
Table 2: Showing median values, standard deviation and statistical significance at 5% using ANOVA for TNF - α level in GCF

Click here to view
Table 3: Showing median values, standard deviation and statistical significance at 5% using ANOVA for TNF - α level in serum

Click here to view
Table 4: Spearman's correlation coefficient (r) between the levels of TNF - α in GCF and serum in each group

Click here to view




Statistical inference for TNF-α in GCF and serum by using Dunn's multiple comparison for pair wise comparison indicated that, the medians significantly differ at 5% level of significance between group I and group II and between groups I and group III, as the absolute difference between the samples mean rank exceeds the LSD. However, between the group II and group III, the absolute difference between the samples mean rank does not exceed the LSD, inferring that the medians do not differ at 5% level of significance.

Spearman's correlation coefficient between the level of TNF-α, in GCF and serum compares the mean and median levels of TNF-α in all 3 groups. Group II showed that the levels of TNF-α in GCF was about 5.06 times higher than the serum levels, group III showed that the median levels of TNF-α in GCF was about 7.39 times higher than the serum levels. However, when Spearman's test was done to assess the relationship between the level of TNF-α in GCF and serum in each group at 5% level of significance, there was absolutely no correlation between the levels of TNF-α in GCF and serum [Table 4].


   Discussion Top


Periodontitis is characterized by inflammatory destruction of connective tissue, loss of periodontal attachment and resorption of alveolar bone. Cytokine synthesis and release by cells in the affected tissues possesses bioactivities, which are consistent with a causative or contributory role in the destruction of bone and connective tissue in periodontitis. [11] Several cytokines have been detected in GCF and in gingival tissues of patients with periodontitis, reflecting the possibility of evaluating the contents of GCF as "Indicators" or "Markers" of periodontal disease. [10]

Evaluation of the contents of GCF is a promising, non-invasive method for determining tissue changes in periodontium. [9] The suitability of using cytokine TNF-α in GCF as a possible indicator of periodontal disease was first assessed by Rossomando. [10] Till date, there are not many studies comparing the levels of TNF-α in GCF and serum and its relationship to periodontal disease. Hence, an attempt is made to estimate the level of TNF-α in GCF and serum and to explore its relationship to periodontal disease. In the present study, a total of 60 subjects were examined. The subjects were grouped into control, gingivitis and periodonititis based on Ramfjord's Periodontal Disease Index and radiographic evidence of bone loss.

In this study, we found a wide range of TNF-α in the tested samples. Hence, median values of the level of TNF-α, was taken into consideration for the statistical analysis instead of mean values.

We also found significant difference in TNF-α levels in GCF between the 3 groups. Highest levels belonged to gingivitis group. Higher levels in group II as compared to groups I and III, indicates a positive correlation with inflammatory changes in gingivitis. [Table 1]

This is in similar to the study by Rossomando, [10] who showed that 62% of sites containing TNF-α had gingival index of 1. However, the results are inconsistent with Heasman, [12] who found that the TNF-α levels in GCF did not change in experimental gingivitis, which could be attributed to different experimental design and difference in the sensitivity of the ELISA kit, used for the detection of TNF-α. The sensitivity of the ELISA kit used in the previous study was 2ng/ml as compared to the ELISA kit with the sensitivity of 4pg/ml in our study.

Increased level of TNF-α in GCF in group II subjects is related with an inflammation. High numbers of inflammatory cells in the connective tissue and gingival crevice can lead to the release of TNF-α upon stimulation by the bacterial products. [2]

It was not surprising that small amount of TNF-α could also be detected in sites of group I subjects. Stashenko and Jandinski [11] demonstrated by immunofluroscent technique that, TNF-α positive staining cells were also present in normal gingival tissues, but are much lesser than that found in the inflamed tissues. The role of cytokines in normal sites may be related to the physiological activities. The presence of low number of macrophages and mononuclear cells in the gingival tissues and neutrophils in GCF in clinically normal tissues could also account for the presence of TNF-α in GCF in group I subjects. [2]

The median level of TNF-α in GCF in group III subjects was 86 pg/ml. Comparison of the median levels of TNF-α in GCF between groups I and III showed results to be statistically significant inferring a positive correlation with periodontitis. Stashenko and Jandinski [11] have reported that, there were increased levels of TNF-α in gingival tissues of periodontitis patients. These data suggest that TNF-α is related with the inflammatory condition of the periodontium. TNF-α may be synthesized and secreted by the local periodontal connective tissue cells, such as fibroblasts and endothelial cells or by infiltrating leukocytes i.e. mononuclear cells, macrophages and neutrophils.

When the median levels of TNF-α between groups II and III were compared, the results did not show any statistically significant difference. Absence of detectable levels of TNF-α in GCF in some sites of group III subjects could explain the quiescent nature of the pocket with no active inflammatory process. [5]

The median levels of TNF-α in serum in group II and III subjects were higher than that of group I subjects. This is similar to the results showed by Meyle J, [8] who reported elevated levels of TNF-α in serum of periodontitis patients as compared to healthy controls. However, the results are inconsistent with the data presented by Shapira, Van Dyke, [13] who showed no increase in serum levels of TNF-α in LJP patients. Criteria for including the periodontitis patients in disease groups are varied and this might explain the variability of results demonstrating the correlation between the TNF-α and periodontitis.

From the above observations, one can conclude that the local inflammatory changes that have resulted in elevated levels of TNF-α in GCF might have contributed to the increased levels of TNF-α in serum i.e., systemic "Spill" of cytokine via the circulation as reported by Offenbacher. [5] The absence of detectable levels of TNF-α in GCF in some sites argues against the purely systemic origin for the cytokine.


   Conclusion Top


These observations suggest a positive association between periodontal disease and increased levels of TNF-α in GCF and serum. It can be concluded that there is a possibility of using the estimation of TNF-α in GCF as a "marker" of periodontal disease. However, a cross-sectional study such as this cannot relate the presence or absence of TNF-α in GCF to disease activity, but it remains a possibility that the absence or low levels of this cytokine in GCF might indicate a stable lesion and elevated levels might indicate an active site; but only longitudinal studies taking into account, the disease "activity" and "inactivity" could suggest the possibility of using TNF- α in GCF as a biochemical "Marker" of periodontal disease.

 
   References Top

1.Ranney RR. Immunologic mechanisms of pathogenesis in periodontal diseases: An assessment. J Periodont Res 1991;26:243-54.  Back to cited text no. 1
[PUBMED]    
2.Page RC. The role of inflammatory mediators in the pathogenesis of periodontal disease. J Periodont Res 1991;26:230-42.  Back to cited text no. 2
[PUBMED]    
3.Jan Vilaek, Lee TH. Tumor Necrosis Factor -New insights into the molecular mechanisms of its multiple actions. J Biol Chem 1991;266:7313-6.  Back to cited text no. 3
    
4.Vassalli P. The Pathophysiology of Tumor Necrosis Factors. Ann Rev Immunol 1992;10:411-52   Back to cited text no. 4
    
5.Offenbacher S. Periodontal Diseases: Pathogenesis. Ann Periodontol 1996;1:821-78.  Back to cited text no. 5
[PUBMED]    
6.Van Dyke TE, Lester MA, Shapira L. The role of the host response in periodontal disease progression: Implications for future treatment strategies. J Periodontol 1993;64:792-806.  Back to cited text no. 6
    
7.Bertolini DR, Glenn EN, Bringman TS, Smith DD, Mundy GR. Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors: Nature 1986;319:516-8.  Back to cited text no. 7
    
8.Meyle J. Neutrophil chemotaxis and serum concentration of tumornecrosis-factor-α. J Periodontol Res 1993;28:491-3.  Back to cited text no. 8
[PUBMED]    
9.Lamster IB. The host response in gingival crevicular fluid: Potential applications in periodontitis clinical trials. J periodontol 1992;63:1117-23.  Back to cited text no. 9
    
10.Rossomando EF, Kennedy JE, Hadjimichael J . Tumour necrosis factor alpha in gingival crevicular fluid as a possible indicator of periodontal disease in humans. Arch Oral Biol 1990;35:431-4.  Back to cited text no. 10
    
11.Stashenko P, Jandinski JJ, Fujiyoshi P, Rynar J, Socransky SS. Tissue Levels of Bone Resorptive Cytokines in Periodontal Disease. J Periodontol 1991;62:504-9.  Back to cited text no. 11
    
12.Heasman PA, Collins JG, Offenbacher S. Changes in crevicular fluid levels of interleukin-1β, leukotriene B 4 , prostaglandin E 2 , thromboxane B 2 and tumour necrosis factor α in experimental gingivitis in humans. J Periodontol Res 1993;28:241-7.  Back to cited text no. 12
    
13.Shapira L, Warbington M, Van Dyke TE. TNFα and IL-1β in serum of LJP patients with normal and defective neutrophil chemotaxis. J Periodontol Res 1994;29:371-3.  Back to cited text no. 13
    

Top
Correspondence Address:
Kalpana Gokul
Department of Periodontics, Priyadarshini Dental College & Hospital, Chennai
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.102221

Rights and Permissions



 
 
    Tables

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

This article has been cited by
1 Evaluation of the salivary levels of visfatin, chemerin, and progranulin in periodontal inflammation
Erkan Özcan,N. Isil Saygun,Muhittin A. Serdar,Nezahat Kurt
Clinical Oral Investigations. 2014;
[Pubmed] | [DOI]
2 Role of cytokines in development of pre-eclampsia associated with periodontal disease - Cohort Study
Ashok Kumar,Nargis Begum,Sudha Prasad,Arundeep K. Lamba,Mahesh Verma,Sarita Agarwal,Shashi Sharma
Journal of Clinical Periodontology. 2014; 41(4): 357
[Pubmed] | [DOI]



 

Top
 
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  
 


    Abstract
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Tables

 Article Access Statistics
    Viewed2720    
    Printed82    
    Emailed0    
    PDF Downloaded176    
    Comments [Add]    
    Cited by others 2    

Recommend this journal