Association between interleukin-6, C-Reactive protein and periodontal inflamed surface area : a cross- Sectional investigation

Introduction

Periodontal disease is a common, complex multifactorial disease characterized by the destruction of periodontal tissues and loss of connective tissue attachment. The clinical findings in periodontitis have emphasized the local nature of the inflammation and tissue destruction within the oral cavity. The acute-phase response is one of the primary defense mechanisms to restore homeostasis and to remove the irritant. It is a non specific process that occurs as initial host response to noxious stimuli¹ which leads to systemic changes including the hepatic release of various plasma proteins known as acute phase proteins. There have been extensive clinical studies showing significant increase in systemic levels of various acute-phase reactants in patients with periodontitis compared with periodontally healthy individuals.²

Periodontitis is emerging as a risk factor for broad range of systemic conditions like cardiovascular diseases, diabetes, stroke, pneumonia, and adverse pregnancy outcomes. Chronic low grade inflammation is considered as a conceivable etiologic mechanism linking periodontal diseases and systemic diseases.^3,4 Many acute phase reactants have been shown to play a crucial role in the linkage between periodontitis and systemic disease markers like C-Reactive Protein (CRP).

C-Reactive Protein is an extremely sensitive and non-specific acute-phase marker for inflammation that is produced in response to many forms of injuries, systemic diseases, oral and periodontal disease. CRP has emerged as an interesting novel and potentially clinically useful marker for increased cardiovascular risk. CRP is associated with endothelial cell damage and has been viewed as marker of low grade vascular inflammation.⁵ Recent studies showed that CRP levels are increased in periodontal disease alone and also when it was associated with systemic diseases.⁶ Release of CRP is regulated by various factors. Plasma levels of CRP are known to be regulated at the transcription level by Interleukin-6 (IL-6).⁷

IL-6 is an important cytokine involved in regulation of host response. It plays an important role in B-cell differentiation and is synergistic with IL-1β to cause bone resorption. Several studies have shown increased level of IL-6 in both GCF and serum of periodontitis patients.⁸

The biological model for the credibility of periodontitis as a risk factor for other diseases holds that the inflammatory burden is caused by periodontitis by eliciting systemic inflammatory responses.⁹ Following this biological model, the larger the amount of inflamed periodontal tissue is, the larger the chances are of periodontitis eliciting systemic inflammatory responses. Therefore, any classification of periodontitis as a risk factor for other diseases should quantify the amount of inflamed periodontal tissue in order to quantify the inflammatory burden.

For this purpose, a new measure Periodontal Inflamed Surface Area (PISA) was developed.¹⁰ PISA reflects surface area of bleeding pocket epithelium in square millimeters and can be calculated using conventional clinical attachment levels (CAL), recession and bleeding on probing. In the light of the above mentioned facts this study is designed to assess serum and GCF levels of CRP and IL-6 and correlate these with PISA in patients affected by chronic periodontitis.

Materials and Methods

Study population: The study populations consisted of 45 subjects belonging to both the sexes. Subjects were selected from the outpatient department of M R Ambedkar Dental College and Hospital, Bangalore after the permission was obtained from the ethical committee. Written informed consent was obtained and patients fulfilling the below mentioned criteria were recruited.

Inclusion Criteria:

1. Patients between 35-55 years of age.

2. Patients with chronic generalized periodontitis 

3. Subjects with ≥20 teeth excluding the third molars, with at least 8 teeth showing ≥5mm of clinical attachment loss and ≥5mm of probing pocket depth.

Exclusion criteria:

1. Patients with a historyof increased bleeding tendencies.

2. History of underlying systemic disease, which affects the periodontal condition.

3. History of tobacco use in any form.

4. Pregnancy and lactating female.

5. Patients on any systemic antibiotics, NSAIDs, steroid therapy, statins or any other drugs which affect the periodontal condition.

6. Patients who have undergone any periodontal therapy in past 2 years.

Periodontal parameters recorded: After recording the demographic data, the following periodontal parameters were recorded: Bleeding index (Ainamo and Bay), Plaque index (Sillness and Loe),Probing depths and clinical attachment level (with CEJ as reference) using UNC-15 probe at six sites per tooth. Periodontal Inflamed surface area (PISA)was recorded as suggested by Huojel.

Estimation of CRP and IL-6 in serum and GCF: 3µl GCF was collected, from the deepest probing pocket depth site. GCF was collected by extra crevicular method using color coded, volumetric, calibrated microcapillary pipettes with 0.5 µl range. The pipettes contaminated with blood or saliva were discarded.2ml of blood was collected from antecubital fossa by venipuncture in EDTA coated tubes following aseptic precautions and collected sample was then centrifuged to separate serum from the sample. The samples of GCF and serum were transferred into Eppendorf tubes and stored at -70oC until analyzed for IL-6 and CRP. C- Reactive Protein (CRP) and IL-6 levels were estimated by Enzyme linked immunosorbent assay (ELISA) method using RayBio® kits.

Statistical Analysis: Descriptive statistical analysis was carried out in the present study. Results on continuous measurements were presented as mean ±SD and results on categorical measurements were presented in number(%). Significance was assessed at 5% level of significance. Pearson’s correlation has been used to find the relationship between GCF and serum concentrations of CRP, IL-6 and PISA. Multiple regression analysis was conducted with PISA as the dependent factor. The statistical significance was considered at p<0.05.

Discussion

Periodontitis is an immunoinflammatory disease which results in the loss of connective tissue attachment of a tooth. Gram-negative anaerobes are present in large numbers in subgingival dental plaque in periodontal pockets. Endotoxin derived from these bacteria activate signals in both the innate and adaptive immune systems leading to production of cytokines which coordinate the local and systemic inflammatory response.¹¹

There is ample evidence to prove the effect of periodontal inflammation on systemic disease. Pro-inflammatory cytokines originating at the site of local pathology activate hepatocytes to produce acute phase proteins including CRP.¹² Epidemiological studies have shown that levels of acute phase proteins including CRP are increased in otherwise healthy adults with poor periodontal status^13,14 and CRP is currently regarded as a biomarker of systemic inflammation. The reason for the interest in plasma levels of CRP in periodontitis lies in the fact that epidemiological research indicates that periodontitis is associated with cardiovascular disease. A major drawback of the studies published on periodontal inflammationsystemic disease interaction is the lack of a tool that adequately assesses the inflammatory burden of periodontitis. The traditional clinical parameters such as probing depth, gingival bleeding on probing, GCF flow, and radiographic levels of alveolar bone are not accurate in assessing the inflammatory burden. Therefore, a new measure of periodontitis, the periodontal inflamed surface area (PISA) has been developed. PISA reflects the surface area of bleeding pocket epithelium. PISA quantifies the amount of inflamed periodontal tissue, thereby quantifying the inflammatory burden posed by periodontitis.

CRP is closely linked with IL-6, an important pro-inflammatory cytokine, which acts as the principal inducer of CRP synthesis by the liver.¹⁵ Interleukin-6 is a multifunctional protein that stimulates immunoglobulin secretion by human B-lymphocytes, activates T-cells, stimulates the synthesis and secretion of acute phase proteins by hepatocytes, and activates the complement cascade. IL-6 has also been shown to play a major role in the terminal differentiation of B-lymphocytes to plasma cells, which are predominant inflammatory cells in tissues involved in established and advanced periodontal disease.

The above mentioned facts lead to the fact that periodontal inflammation can influence the systemic health as proved by the increase in serum CRP levels. Hence the present study was conducted to evaluate systemic inflammatory burden posed by periodontal inflammation. The systemic inflammatory burden was assessed using serum CRP and serum IL-6 levels. Extent of periodontal inflammation was calculated using PISA, GCF CRP and GCF IL-6 levels.

The study population for the present study included 45 systemically healthy patients with chronic generalized periodontitis. Demographic data and periodontal parameters were recorded for all the patients. Levels of CRP and IL-6 were measured in the serum and GCF using ELISA.

The present study showed that there was positive correlation between PISA and all the periodontal parameters, i.e. PI, BI, CI and mean CAL. BI and CAL showed a highly significant correlation with PISA. This is suggestive of an increase in PISA value with increase in bleeding index and mean CAL. This positive correlation of the periodontal parameter with PISA clearly indicates the validity of PISA index as a measure of inflammation.

The determination of presence of inflammatory products in GCF and serum might be useful for evaluating the periodontal disease status and systemic burden it poses. The results of present study showed a level of 4.51+1.36 mg/L of CRP in the serum of the study population. This is in accordance with various studies that have demonstrated higher levels of serum CRP in subjects with periodontal disease.^16,17 The mean CAL for the study population was 4.52 + 0.59 mm and the findings agree with the observation of Slade et al.¹⁸ who reported elevated serum CRP in individuals with extensive periodontal disease (>30% sites with pockets > 4mm). However, many studies have contradicted the presence of elevated levels of CRP due to periodontitis.¹⁹

Evidence has shown that periodontitis is associated with a significant increased risk of sustained high (>3 mg/L) CRP levels which can lead to increased risk of future cardiovascular events.²⁰ Our results suggested that there was a positive correlation between PISA and serum CRP levels. Our results are in agreement with previous studies that have shown that the systemic CRP levels increased with increasing severity of periodontal disease.^21,22

GCF is known as the window to the changes occurring in the periodontal tissues. Hence, evaluating the constituents of GCF is considered as an important diagnostic tool in periodontal disease.GCF-CRP levels were measured in our study and the results showed that mean GCF-CRP level was 5.36 + 3.35 mg/L. There is evidence to suggest that there is local production of CRP in gingival tissues²³ and this justifies the increase in GCF-CRP levels in periodontitis patients. Most of the studies in periodontal literature have evaluated serum CRP levels and a very few have examined GCF-CRP levels.^24,25

Our study showed that there was a positive correlation between PISA and GCF-CRP levels. This is in agreement with other studies^26,27 which showed that the levels of GCF-CRP increased proportionately with increase in severity of the periodontal disease.It has been shown that plasma level of CRP is mainly regulated at the transcriptional level by IL-67. It has been seen that the local and systemic levels of IL-6 are increased in periodontitis13 and this may affect CRP production. In the present study, we found a positive correlation between the levels of CRP and IL-6 in the GCF and the serum of the patients. To the best of our knowledge there is no study published where IL-6 and CRP are correlated in GCF and serum.

The present study showed a mean GCF-IL6 concentration of 6.45 + 4.74 pg/ml. The results of our study also showed a positive correlation between PISA and GCF-IL6. Various studies have shown that periodontitis is associated with increase in levels of IL-6.^28,29 Atilla and Kutukculer³⁰ detected higher GCF IL- 6 levels in sites with gingivitis than the healthy sites. Also, Lee et al.³¹ reported higher GCF IL-6 levels in active than in inactive sites. On the contrary, Bozkurt et al.³² suggested that in patients with adult periodontitis no correlation between GCF IL-6 levels and clinical parameters was found.

The mean serum IL-6 concentration of 9.34 + 5.96 pg/ml was seen in the present study. A significant positive correlation between PISA and serum IL-6 was seen. Previous studies have found that serum IL-6 is related to periodontal parameters like BOP, PD, and CAL.³³

There is negligible literature available on association between PISA and inflammatory markers. A study done in 2015 by Govindrajan et al³⁴ suggested a positive correlation between PISA and IL-1 alpha levels in GCF. Henri Susan to³⁵ analyzed the validity of PISA index by showing that the periodontitis severity and CRP can predict Hb1Ac in healthy individuals and individuals treated for Diabetes Mellitus type 2. Nesse et al, 2009 showed a dose- response relationship between periodontal surface area and HbA1c in type 2 Diabetics.³⁶

To conclude, the results of our study suggest that there was a positive correlation between PISA and inflammatory markers both in serum and GCF. PISA could probably be used to correlate local inflammatory burden with systemic inflammatory burden. The results should be cautiously interpreted since the study has certain limitations. It was a cross-sectional study with no control group. Comparison with a control group consisting of healthy individuals could have given wider interpretations. The sample size was not large enough to draw conclusions. Another limitation was that no intervention was done which could have led to a better understanding of the effect of periodontal treatment on systemic inflammatory burden. Larger sample sized randomized controlled trials are required to confirm the hypothesis of this study.