The role of interleukin-6 in pathogenesis of chronic periapical lesions

Summary Introduction Cytokine network plays an important role in pathogenesis of chronic periapical lesions. The aim of this study was to determine the concentration of interleukin-6 (IL-6) in tissue homogenates of human periapical lesions and correlate its levels with symptomatology and size of the lesions. Materials and Methods 93 samples of chronic periapical lesions were obtained after extraction of teeth. Samples were divided according to the clinical presentation as symptomatic and asymptomatic, and according to the size as large and small. The concentration of IL-6 was analyzed using ELISA. Results Statistically significant difference in IL-6 concentration was observed in symptomatic lesions compared to asymptomatic (p<0.001). Analysis showed statistically higher concentration in large symptomatic lesions compared to large asymptomatic lesions (p<0.001), and in small symptomatic lesions compared to small asymptomatic (p<0.05). Higher production of IL-6 was observed in large lesions compared to small but this difference was not statistically significant. Conclusion Higher concentration of IL-6 in lesions with expressed clinical symptoms as well as in large lesions indicates that IL-6 is an important factor responsible for the progression of lesions and bone resorption.


INTRODUCTION
Periapical lesions are inflammatory disorders that develop as a result of an immune response to continuous antigen stimulation from the root canal. Their development and progression to chronic lesions with concomitant bone resorption occur as a result of inability of host defense mechanisms to remove infection [1]. Chronic periapical lesions represent inflamed granulation tissue infiltrated by different inflammatory cells, which produce a variety of mediators. Cytokine network plays an important role in the regulation of non-specific and specific immune responses. Many studies have demonstrated the production of various cytokines in the periapical lesions [2,3].
Interleukin 6 (IL-6) is a cytokine that influences the antigen-specific immune responses and inflammatory reactions, and has the role of proinflammatory and antiinflammatory cytokine [4]. IL-6 is the "myokin", a cytokine produced in the muscles in response to contraction. It increases significantly with exercise, and prior to the occurrence of other cytokines in the bloodstream. IL-6 production is proven in human periapical lesions [5] and in marginal inflamation of periodontal tissues [6]. The literature data suggests that IL-6 is multifunctional cytokine produced by several types of immune cell-monocytes, macrophage, Th-2 cells, activated B cells, and polymorphonuclear cells [7]. Production of IL-6 is carried out under the influence of IL-1, TNF-α and INF-γ, however, it also regulates the secretion of IL-1 and is opposed by some of its effects. IL-6 is secreted by osteoblasts during stimulation of osteoclast formation. IL-6 stimulates the formation of osteoclast precursors and increases the number of osteoclasts in vivo, leading to systematic bone resorption [8]. Along with TNF-α and IL-1, it belongs to the group of major proinflammatory cytokines. Numerous data indicate that IL-6 also has anti-inflammatory activity. The antiinflammatory role of IL-6 is mediated through the inhibitory effects of TNF-α and IL-1 [9].
The aim of this study was to determine the concentration of interleukin-6 (IL-6) in tissue homogenates of human periapical lesions and correlate its levels with symptomatology and size of the lesions.

MATERIALS AND METHODS
The study included 93 patients from the Clinic of Dentistry, Nis, who were diagnosed with chronic periapical lesions using clinical and radiographic methods. The study was approved by the Ethical Committee of the Medical Faculty, University of Nis, Serbia (no. 01-2066-5). Periapical lesions were collected from teeth that were determined as non-salvageable and indicated for extraction.
From each patient, dental history including symptomatology and medications was collected and clinical exam was performed. Other inclusion criteria were healthy patients not suffering from acute or chronic diseases that could lead to immunodeficiency, and who were not taking antibiotics and anti-inflammatory medications in the last two months.
Only teeth with periapical lesions that did not show moderate or severe form of marginal periodontitis were included in the study. According to subjective symptoms of patients, lesions were divided into the two groups: symptomatic and asymptomatic. Clinically symptomatic lesions were characterized by swelling, pain, discomfort when chewing or sensitivity to percussion and palpation whereas asymptomatic lesions showed no symptoms. The size of periapical lesions was measured in millimeters using a ruler and divided into the two groups: small (≤5 mm) and large (≥6 mm) ( Table 1). Since periapical lesions contain granulomatous inflammatory tissue that replaces normal bone there was no equivalent tissue that could be used as negative control.
Before administering local anaesthetics, teeth, gingiva and mucosa around the tooth were cleaned using 0.12% chlorhexidine and the patient rinsed mouth with 0.12% chlorhexidine for 30 seconds. Samples of periapical lesions removed from the root apex were collected immediately after the extraction using sterile scalpel, then washed in sterile saline solution, dried with sterile cotton, placed in a sterile plastic Eppendorf tubes and frozen at -70°C. Using teflon crusher in an iced phosphate buffer at pH 7.4, samples were homogenized with volume adapted to weight of the tissue obtaining the final concentration of 10%. Larger debris was sedimented by centrifugation at 1400 rpm for 1 minute at -40°C. The supernatant was frozen at -70°C until further analysis was performed.
The concentration of IL-6 was measured using ELISA test (R&D Systems Inc. Minneapolis, USA) according to the manufacturer's instructions. The sensitivity of ELISA test for IL-6 was from 0.7 pg/ml, and the concentration of cytokine was analyzed in relation to the size and symptomatology of periapical lesions. Statistical analysis was performed using the Mann-Whitney Rank Sum test in the software Sigmastat and Origin. The results were expressed as mean ± standard deviation. P<0.05 was considered statistically significant.

RESULTS
All tissue homogenates of periapical lesions showed significant concentration of IL-6 cytokine. Figure 1 shows the concentration of IL-6 in the tissue homogenates of all samples that were analyzed with respect to the size and symptomatology. In symptomatic lesions average concentration of IL-6 was 975.51 pg/ml, while in the group of asymptomatic lesions the average value was 212.04 pg/ml. Analysis of the concentration showed that there was significantly higher concentration of IL-6 in symptomatic lesions (p<0.001). In the group of large lesions, average concentrations of IL-6 amounted to 687.14 pg/ml, while in the group of small lesions average value was 470.92 pg/ml. The difference was observed but not statistically significant. Table 2 shows the concentration of IL-6 within the group of symptomatic and asymptomatic lesions, and the concentration of cytokines in each of the groups were analyzed with respect to the size. The average concentration of IL-6 in the large symptomatic lesions amounted to 1162.40 pg/ml, while in the small symptomatic lesions it was 759.11 pg/ml. A statistically significant difference in the concentrations of IL-6 was not observed in the symptomatic lesions in relation to the size. In the group of asymptomatic lesions, statistically significant differ-    Figure 2 shows the concentration of IL-6 in the groups of large and small lesions where the statistical significance was analyzed in relation to symptomatology. The average concentration of IL-6 in large symptomatic lesions amounted to 1162.40 pg/ml, while in large asymptomatic lesions it was 189.24 pg/ml. The difference was statistically significant (p<0.001). The analysis showed significantly higher concentration in small symptomatic (759.11 pg/ml) compared to small asymptomatic lesions (232.86 pg/ml) (p<0.05).

DISCUSSION
Periapical lesions develop as a result of persistent inflammatory response induced by prolonged exposure of periapical tissue to root canal microorganisms, causing an immune reaction. In this local defense mechanism different inflammatory mediators play a complex and central role in the regulation of immune response. While proinflammatory cytokines, such as IL-1, IL-6, TNF-α, TNF-β, chemokines and Th1 cytokines, promote inflammation in the periapical tissues and activate osteoclastic bone resorption [10,11], the role of antiinflammatory cytokines is important for suppression of inflammatory processes and repair processes within the periapical lesions [2,3,12].
In the early inflammatory cascade IL-1 and TNF-α induce production of IL-6. IL-6 has many molecular forms, and each molecule has a different function if secreted by various cells in different situations. The finding that polymorphonuclear cells in the periapical tissues produce IL-6, which was specified by the Euler et al. [13], suggest that IL-6 can contribute to the tissue injury at the site of inflammation. IL-6 is an integral mediator of the acute phase response to injury and infection that stimulate expression of acute phase protein [14]. Examination of various cytokines, such as TNF-α, IL-6, IL-3, GM-CSF, IL-11, IL-17, IL-18, in human and animal models have demonstrated their potential role in the pathogenesis of osteo-lytic diseases [2,15]. It has been shown that inflammatory cytokines IL-6 and TNF-α have the capacity to trigger osteoclastic bone resorption and their role can express synergistically with IL-1, causing activation or osteoclast differentiation and production, as well as secretion of prostaglandins by numerous cell types, including fibroblasts and osteoblasts [2]. Several authors have published the expression of IL-6 production in human periapical granulomas and cysts. Results of our study showed the presence of IL-6 in all tissue samples of periapical lesions that is in accordance with data from the literature [9,16]. Studies have shown that the level of IL-6 is significantly increased in the infection and pain conditions. In the study of De Jongh et al. [17] important role is attributed to IL-6 in the pathophysiology of pain. Due to this fact, in our study we analyzed correlation between levels of IL-6 and symptomatology. The results of our investigations indicated statistically significant difference in IL-6 production in the symptomatic compared to asymptomatic lesions, while the average concentration of cytokines was higher in the large lesions compared to small, but the difference was not statistically significant.
The study of Gazivoda et al. [18] showed higher production of IL-6 in symptomatic and large lesions primarily emphasizing its proinflammatory aspect. However, experiments on IL-6-deficient mice showed conflicting results, indicating protective effect of this cytokine to bone destruction [14]. These differences may be explained by the fact that IL-6 has both proinflammatory and antiinflammatory role and its final effect depends on the target cells and coordination with additional cytokines.
Inflammatory reaction in patients with apical periodontitis is not limited to periradicular region [19]. It is known that dental infection can have negative impact on general health in patients with risk [20]. Blood analysis in patients after endodontic treatment of teeth with apical periodontitis showed the presence of bacteria that originate from root canal [21]. Spreading oral bacteria through the bloodstream is not the only way for mediation-targeted effects of oral focal infections. More significant effect on distant tissues and organs function may be induced by certain cytokines. Although the majority of members of the cytokine superfamily exhibits short-term effects, IL-1, IL-6 and TNF have been shown that locally produced within the tissues of periapical granulomas, can be carried by the bloodstream to distant places. These cytokines can cause acute phase response, which includes fever, increased erythrocyte sedimentation rate, and change in serum proteins synthesized by hepatocytes. Some authors examined elevated levels of acute phase protein in patients with chronic periapical granulomas. Their level was lowered to the normal value after the surgical removal of lesions by apicoectomy [10,22].

CONCLUSIONS
High concentration of IL-6 in lesions with expressed clinical symptoms, as well as in large lesions, indicate its important pro-inflammatory activity and key role of