Radiographic Features and Inflammatory Cytokine Networks Produced by Macrophages Stimulated with Endodontic Content

Radiographic Features and Inflammatory Cytokine Networks Produced by Macrophages Stimulated with Endodontic Content

Clinical Research Correlation between Clinical/Radiographic Features and Inflammatory Cytokine Networks Produced by Macrophages Stimulated with Endod...

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Clinical Research

Correlation between Clinical/Radiographic Features and Inflammatory Cytokine Networks Produced by Macrophages Stimulated with Endodontic Content Frederico C. Martinho, DDS, MSc,* Wanderson M.M. Chiesa, DDS, MSc,* Fabio R.M. Leite, DDS, MSc, PhD,† Joni A. Cirelli, DDS, MSc, PhD,‡ and Brenda P.F.A. Gomes, DDS, MSc, PhD* Abstract Introduction: Macrophages are highly activated by endodontic contents. This study investigated the correlation between different clinical signs/symptoms and radiographic features according to the levels of interleukin (IL)-1b, tumor necrosis factor a (TNF-a), IL-6, IL-10, prostaglandin E2 (PGE2), and their networks produced by endodontic content–stimulated macrophages collected from primary endodontic infection with apical periodontitis (PEIAP). Methods: Samples were taken from 21 root canals with PEIAP by using paper points. The presence of exudate (EX), pain on palpation (POP), tenderness to percussion (TTP), and the size of the radiographic lesion (SRL) were recorded. Polymerase chain reaction (16S rDNA) was used for bacterial detection and limulus amebocyte lysate (LAL) assay for endotoxin measurement. Raw 264.7 macrophages were stimulated with bacterial contents during 24 hs. The amounts of IL-1b, TNF-a, IL-6, IL-10 and PGE2 were measured by enzyme-linked immunosorbent assay. Log-based data were correlated by multiple logistic regression (P < .05). Results: Bacteria and endotoxin were detected in 100% of the samples. IL-6 and TNF-a were positively correlated with SRL and EX, respectively (P < .05). Clinical signs/symptoms and radiographic findings were set as dependent variables for EX-positive correlations between PGE2, IL-1b, and TNF-a (P < .05), whereas IL-6 and PGE2 were positively correlated to each other in POP but negatively correlated in SRL (P < .05). When POP and TTP-POP were set as dependent variables, different cytokine networks were found. Conclusions: Our findings suggest different roles for each cytokine in the development of apical periodontitis, whose effects of overlapping networks

depend on the signs/symptoms and radiographic features found in endodontic infection. (J Endod 2012;38:740–745)

Key Words Bacteria, cytokines, endotoxin, macrophages, root canal

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ndodontic diseases are well recognized as a result of the interaction between host immune response and pathogenic bacteria present in the root canals (1, 2). Infected root canals act as a source of pathogenic species, virulence factors, and inflammatory mediators that spread into apical tissue, creating and sustaining a chronic inflammatory burden (3). The inflammatory process is initiated and maintained by the emergence of a network of chemokines (eg, interleukin [IL]-8) and proinflammatory (eg, tumor necrosis factor a [TNF-a], IL-1b, and IL-6]) and anti-inflammatory mediators (eg, IL-10, IL-1 antagonism, and IL-4) that play distinct or shared biological activities (4). TNF-a stimulates the production of collagenase, prostaglandin E2 (PGE2), chemoand cytokines, cellular adhesion molecules, and bone resorption–related factors (5, 6). IL-6 acts as a proinflammatory cytokine during periodontitis and stimulates osteoclastic differentiation and bone resorption in chronic inflammatory periodontitis (7, 8). PGE2 can induce or repress IL-6 and vice versa (9). Conversely, IL-10, which is produced by both innate and adaptive immune cells, controls and suppresses the inflammation in order to down-regulate the adaptive immune reaction and minimize tissue destruction in response to microbial challenge (10). This cytokine can also inhibit the expression of several proinflammatory cytokines (11, 12). Macrophage cells, which predominate in the inflammatory tissue of the periapical lesions, are considered one of the main sources of inflammatory cytokines (13). It has long been known that a primary endodontic infection has a polymicrobial etiology caused by both gram-positive and gram-negative anaerobic bacteria (14). Lipopolysaccharides (LPSs, known as endotoxins), present on the outer layers of gram-negative bacterial cell walls (15), are one of the most potent stimuli for macrophages in the release of IL-1b, TNF-a, IL-6, PGE2, and IL-10 (16, 17). IL-1b and TNF-a have been detected in periapical tissues (18–21) and root canal EXs (20, 22, 23). Particularly, IL-1b has been correlated with clinical signs/symptoms

From the *Department of Restorative Dentistry, Endodontics Division, Piracicaba Dental School, State University of Campinas, Piracicaba, S~ao Paulo, Brazil; Department of Semiology and Clinics, Periodontics Division, Dental School, Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil; and ‡Department of Diagnosis and Oral Surgery, Periodontics Division, Araraquara Dental School, State University of S~ao Paulo, Araraquara, S~ao Paulo, Brazil. Supported by the Brazilian agencies FAPESP (08/57551-0; 08/56425; 10/51113-1; 10/17877-4 10/19136-1; 11/50510-0) and CNPq (302575/2009-0; 150557/ 2011-6). Address requests for reprints to Dr Brenda P.F.A. Gomes, Piracicaba Dental School, State University of Campinas-UNICAMP, Department of Restorative Dentistry, Endodontics Division, Av Limeira 901, Bairro Areiao, Piracicaba, SP, Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.02.021 †

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Clinical Research (18) and greater bone resorption (24). PGE2 has been reported in painful/asymptomatic human dental pulp (25), exudation (26), and periapical tissue (27). Barkhordar et al (28) revealed significant levels of IL-6 in periapical lesion and inflamed pulp tissues. Nevertheless, none of these studies has considered possible networks among all these different cytokines involved in the development of clinical/radiographic features found in endodontic diseases. Therefore, the aim of this study was to investigate the correlation between different clinical signs/symptoms and radiographic features regarding the levels of IL- 1b, TNF-a, IL-6, IL-10, PGE2, and the networks produced by endodontic content-stimulated macrophages collected from a primary root infection with apical periodontitis.

Materials and Methods Patient Selection Twenty-one patients needing endodontic treatment who attended the Piracicaba Dental School, Piracicaba, Brazil, were included in this research. The age of the patients ranged from 13 to 73 years old. Samples were collected from 21 root canals with pulp necrosis, all showing radiographic evidence of apical periodontitis. The selected teeth showed the absence of periodontal pockets more than 3 mm. The following clinical/radiographic features were found and recorded: the presence of exudate (EX), pain on palpation (POP) (9/21), tenderness to percussion–pain on palpation (TTP-POP) (7/21), and the size of the radiographic lesion (SRL) $2 mm (11/21) and <2 mm (10/21). A detailed dental history was obtained from each patient. Those who had received antibiotic treatment during the last 3 months or who had any general disease were excluded. The Human Research Ethics Committee of the Piracicaba Dental School approved the protocol describing the sample collection for this investigation, and all volunteer patients signed an informed consent form. Sampling Procedures All materials used in this study were heat sterilized at 200 C for 4 hours to become apyrogenic. The method for disinfection of the operative field has been previously described (29, 30). Briefly, the teeth were isolated with a rubber dam, with crown and surrounding structures being disinfected with 30% H2O2 for 30 seconds, followed by 2.5% NaOCl for 30 seconds. Next, 5% sodium thiosulphate was used to inactivate the irrigant. The sterility of the external surfaces of the crown was checked by using a swab sample, which was streaked on blood agar plates for aerobic and anaerobic incubation. A 2-stage access cavity preparation was performed by using sterile/ apyrogenic high-speed diamond bur without the use of water spray but under manual irrigation with sterile/apyrogenic saline solution. The first stage was aimed to promote most of the removal of contaminants, whereas in the second stage the access cavity was disinfected according to the protocol described earlier. Sterilization of the internal surface of the access cavity was checked as described previously, and all procedures were performed aseptically. A new sterile and apyrogenic bur was used followed by irrigation of the root canal access with sterile apyrogenic water. The endotoxin sample was obtained by introducing sterile pyrogen-free paper points (size #15; Dentsply-Maillefer, Ballaigues, Switzerland) into the full length of the canal (determined radiographically) and retained in position during 60 seconds. Next, the paper points were immediately placed in a pyrogen-free glass and suspended in 1 mL of limulus amebocyte lysate (LAL) water, from which an aliquot was used for stimulating macrophages, detecting bacteria and quantifying endotoxins as described later. JOE — Volume 38, Number 6, June 2012

Bacterial Detection (Polymerase Chain Reaction 16S rDNA) DNA Extraction. Microbial DNA was extracted from 21 endodontic samples and purified with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA concentration (absorbance at 260 nm) was determined by using a spectrophotometer (Nanodrop 2000; Thermo Scientific, Wilmington, DE). Polymerase Chain Reaction Assay. The polymerase chain reaction (PCR) reaction was performed in a thermocycler (MyCycler; Bio-Rad, Hercules, CA) at a total volume of 25 mL containing 2.5 mL of 10 Taq buffer (1) (MBI Fermentas, Mundolsheim, France), 0.5 mL of dNTP mix (25 mmol/L of each deoxyribonucleoside triphosphate [dATP, dCTP, dGTP, and dTTP]) (MBI Fermentas, Hanover, MD), 1.25 mL of 25 mmol/L MgCl2, 0.25 mL of forward and reverse universal primers (0.2 mmol/L) (Invitrogen, Eugene, OR), 1.5-mL sample DNA (1 mg/50 mL), 1.5 mL Taq DNA polymerase (1 U) (MBI Fermentas), and 17.25 mL nuclease-free water. The ubiquitous primer pair used for bacteria detection was as follows: sense 50 -TCC TAC GGG AGG CAG CAG T-30 and antisense 50 -GGA CTA CCA GGG TAT CTA ATC CTG TT-30 with an amplicon size of 466 bp. The optimized cycling conditions were the following: initial denaturation at 95 C for 10 minutes, 40 cycles at 95 C for 10 seconds and 60 C for 10 seconds, and a final extension step at 72 C for 25 seconds. Determination of Endotoxin Concentration (Turbidimetric Test/LAL Assay) The turbidimetric test (BioWhitaker, Inc, Walkersville, MD) using the LAL technique was used to measure endotoxin concentrations within the root canals. First, as a parameter for the calculation of the amount of endotoxins in root canal samples, a standard curve was plotted with the endotoxins supplied in the kit with known concentration (100 EU/mL) and their dilutions at the final concentrations (ie, 0.01, 0.10, 1, and 10 EU/mL) according to the manufacturer’s instructions. Test Procedure. All reactions were performed in duplicate to validate the test. A 96-well microplate (Corning Costar, Cambridge, MA) was set a heating block at 37 C and maintained at this temperature throughout the assay. Initially, the endotoxin samples were suspended in 1 mL of LAL water supplied with the kit and agitated in vortex for 60 seconds before being serially diluted to 101. Immediately, 100 mL of blank was added according to the standard endotoxin solutions at concentrations of 0.01, 0.10, 1, and 10 EU/mL, with 100 mL of the samples being added to 96-well microplate in duplicate. The test procedure was performed according to the manufacturer’s instructions. The absorbencies of endotoxin were measured individually by means of an enzyme-linked immunosorbent assay (ELISA) plate reader (Ultramark, Bio-Rad) at 340 nm. Calculation of Endotoxin Concentrations. Because the mean absorbance value of the standards was directly proportional to the concentration of endotoxins present in the samples, the endotoxin concentration was determined from the standard curve. Cell Culture and Cytokine Expression Mouse macrophages (RAW 264.7) were cultured in 100-mm culture plates by using DMEM supplemented with 100 IU/mL of penicillin, 100 mg/mL of streptomycin, and 10% heat-inactivated fetal bovine serum and maintained in a humidified atmosphere at 37 C and 5% CO2 up to 90% confluence. All tissue culture reagents were obtained from Invitrogen (Eugene, OR). Macrophages were counted in a Neubauer chamber, with 104 macrophages growing per well after 48 hours in 6-well plates. The macrophages were deinduced by 8-hour incubation Clinical/Radiographic Features and Inflammatory Cytokines

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Clinical Research in culture medium (DMEM) containing only 0.3% fetal bovine serum for cell cycle synchronization and then stimulated with 60 mL of root canal contents during 24 hours in order to quantify the total amounts of IL-1b, IL-6, IL-10, TNFa, and PGE2 released in the culture media. The supernatants were collected and stored at 80 C until protein evaluation.

Messenger RNA Expression The macrophage cell viability was tested in the present study by assessing its capacity to express IL-1b, IL-6, IL-10, TNF-a, PGE2, and messenger RNA (mRNA) after 24 hours of root canal content stimulation. A total of 104 macrophages were grown for 48 hours in each well of the 6-well plates, deinduced by 8-hour incubation in culture medium (DMEM) containing 0.3% fetal bovine serum, and then stimulated with 60 mL of primary infection contents for 24 hours for PGE2 mRNA expression. The total RNA was isolated from cells by using Trizol (Invitrogen) according to the manufacturer’s instructions. Both the quantity and the purity of total RNA were determined with a Biomate 3 spectrophotometer (ThermoSpectronic, Rochester, NY). Complementary DNA was synthesized by reverse transcription of 500 ng of total RNA in which 2.5 mmol/L Oligo (dT)12-18 primers and 1.25 U/mL Moloney murine leukemia virus reverse transcriptase were used in the presence of 3 mmol/L MgCl2, 2 mmol/L dNTPs, and 0.8 U/mL of RNAse inhibitor according to the manufacturer’s protocol (Improm II; Promega, Madison, WI). The PCR reaction was performed with a MyCycler (Bio-Rad) thermocycler using 2 mL of RT reaction product for a 25-mL total volume of PCR reaction mix (GoTaq Flexi, Promega) in the presence of 100 pmol/ mL of each gene’s primers (50 pmol/mL of sense and antisense primers) for IL-1b, IL-6, IL-10, TNFa, PGE2, and GAPDH genes, yielding products of 494, 428, 315, 451, 329, and 418 bp, respectively. The primer pair used for IL-1b (NM031512) was the following: sense 50 -GACCTGTTCTTTGAGGCTGA-30 , antisense 50 -CGTTGCTTGTCTCTCCTTGT-30 ; IL-6 (NM012589), sense 50 -tcaactc catctgcccttcag-30 , antisense 50 -AGGCAGTGGCTGTCAACAACA-30 ; IL-10 (NM012854.2) sense 50 -CCTGCTCTTACTGGCTGGAG-30 , antisense 50 TCTCCCAGGGAATTCAAATG-30 ; TNF-a (NM012675) sense 50 -GGAGAAC AGCAACTCCAGAA-30 , antisense 50 -TCTTTGAGATCCATGCCATT-30 ; PGE2 (NM011198) sense 50 -TGCAACAGCTCAATGACTTCC30 , antisense 50 -GC CCCTCACGGACAATGTAGT-30 ; and GAPDH (NM008084) sense 50 -CACCA TGGAGAAGGCCGGGG-30 , antisense 50 -GACGGACACATTGGGGTAG- 30 . The optimized cycling conditions used were the following: initial denaturation at 95 C for 2 minutes and 35 cycles of 95 C for 1 minute, 58 C for 1 minute, 72 C for 2 minutes, and a final extension step at 72 C for 7 minute in the presence of 1.5 mmol/L MgCl2. For the GAPDH, such conditions were the following: initial denaturation at 95 C for 2 minutes and 25 cycles at 95 C for 1 minute, 52 C for 1 minute, 72 C for 1 minute, and a final extension step at 72 C for 10 minutes in the presence of 1.5 mmol/L MgCl2. PCR products were resolved by electrophoresis on 1.5% (w/v) agarose gels containing ethidium bromide (0.5 mg/mL). The amplified DNA bands were analyzed densitometrically by using a digital imaging capture device (Image Quant 100; GE Healthcare, Sunnyvale, CA) and ImageJ 1.32j software (National Institute of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/). The density of the bands corresponding to PGE2 mRNA in each sample was normalized to the quantity of housekeeping gene GAPDH and expressed as fold change over unstimulated control. Total Protein Levels Released into the Culture Media by Macrophages The total amount of protein released into the culture media after root canal content stimulation was measured by using the Coomassie 742

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(Bradford) Protein Assay kit (Thermo Scientific, Rockford, IL). As a parameter for the calculation of the amount of protein released into the culture media, a standard curve was plotted with bovine serum albumin supplied with the kit at a known concentration (2.0 mg/mL) and then serially diluted (0, 25, 125, 250, 500, 750, 1,000, 1,500, and 2,000 mg/mL). The protein assay was performed according to the manufacturer’s instructions.

Calculation of Protein Concentration The amounts of protein in standard and sample solutions was measured individually by using an ELISA plate reader (Ultramark) at 595 nm. Because this absorbance value was directly proportional to the concentration of protein, the protein concentration in each sample was determined from the standard curve. ELISA The amount of IL-1b, IL-6, IL-10, TNFa, and PGE2 released into the culture media after root canal content stimulation of macrophages was measured by the ELISA Duoset kit (R&D, Minneapolis, MN). A culture of unstimulated macrophage was used as the negative control. Thus, standard, control (unstimulated), and sample solutions were added to the ELISA well plate, which had been precoated with specific monoclonal capture antibody for IL-1b, IL-6, IL-10, TNF-a, or PGE2. After shaking gently for 3 hours at room temperature, the polyclonal anti–IL-1b, anti–IL-6, anti–IL-10, anti–TNF-a, and anti-PGE2 antibodies, conjugated with horseradish peroxidase, were added to the solution and incubated for 1 hour at room temperature. A substrate solution containing hydrogen peroxidase and chromogen was added and allowed to react for 20 minutes. The levels of cytokines were assessed with a micro-ELISA reader at 450 nm and normalized with standard solution. Each densitometric value, expressed as mean  standard deviation, was obtained from 3 independent experiments. Statistical Analysis The data collected were imported into the statistical analysis package Statistica 9.0 (Sta-Soft Inc, Tulsa, OK) for multiple linear regression analysis according to the equation Y’ = a + b1X1 + b2X2 + . + biXi. Cytokine data were log10 decreased. The clinical variables SRL, EX, POP, TTP, and TTP-POP were set as dependent variables. Significance levels were always set at 5% (P < .05). The odds ratio was used to classify the associations between the clinical/radiographic variables and cytokines as positive or negative. Positive associations were those with an average odds ratio >2.0 and negative with an average odds ratio <0.5.

Results No microbial growth was observed in the sterilized samples taken from the external and internal surfaces of the crown, including its surrounding structures, tested before and after entering the pulp chamber.

Bacteria and Endotoxin Detection All root canal samples were positive for bacterial DNA as determined by ubiquitous bacterial primers (21/21). Endotoxin was detected in 100% of the root canals investigated (21/21), with a median value of 7,490 pg/mL, ranging from 27/pg/mL to 289,000 pg/mL. Cytokine mRNA Expression. The macrophage cell viability after 24 hours of root canal content stimulation was confirmed in the present study by its capacity to express IL-1b, IL-6, IL-10, TNF-a, and PGE2. JOE — Volume 38, Number 6, June 2012

Clinical Research Cytokine Production. IL-1b, IL-6, IL-10, TNF-a, and PGE2 were detected in all culture media after stimulation with root canal contents. The median values recorded for each cytokine is in the following: IL-1b (24,835 pg/mL), IL-6 (270,151 pg/mL), IL-10 (39,997 pg/mL), TNF-a (0.2830 pg/mL), and PGE2 (124,530 pg/mL). Individual correlations between each cytokine and the development of clinical signs/symptoms and radiographic findings are shown in Table 1. The SRL was positively correlated with high levels of IL-6 as well as exudation with TNF-a (all P < .05, Table 1). The networks between the different cytokines (positive and negative correlations) investigated after setting the clinical/ radiographic features as dependent variable are shown in Tables 2, 3, 4, and 5.

Discussion Data obtained in the present study revealed that primary endodontic contents can potentially stimulate macrophages to produce a wide variety of cytokines (IL-1b, IL-6, IL-10, PGE2 and TNF-a), which establishes different network interrelationships that are implicated in the development of different patterns of clinical and radiographic features for endodontic diseases. This study was designed to maintain the complex antigenicity of the primarily infected root canal, including the great bacterial and LPS diversity, by stimulating macrophages with endodontic contents sampled from primary endodontic infection followed by measurement of IL-1b, IL-6, IL-10, PGE2, and TNF-a levels. Macrophages, predominantly in the inflammatory periapical tissue (13), are nonspecific defense cells responsible for rapid pathogen recognition and presentation of lymphocytes and other immune cells. Bacteria and their by-products, particularly endotoxins, were detected in 100% of primarily infected root canal samples (30, 31), and, as such, they are potent stimuli for macrophages in the release of IL-1b, TNF-a, IL-6, PGE2, and IL-10 according to previous investigations (16, 17). The overproduction of IL-6 (270,151 pg/mL) and PGE2 (124,530 pg/mL) cytokines particularly found in the present study supports the hyperreactivity of macrophages to stimuli with endodontic contents. In terms of antigenicity, it should be taken into account that other ubiquitous cell-wall components are common to different bacteria, such as the peptidoglycan, which plays a synergistic role in the LPS antigenicity by activating toll-like receptors (TLRs), TLR-2 and TLR-4, respectively (32). This makes endodontic contents even more complex and reactive to macrophages. In the present study, the individual correlation between the different clinical parameters revealed that the SLR was positively correlated with levels of IL-6, thus confirming its role in bone resorption in chronic inflammatory periodontitis (7, 8). Therefore, the presence of

TABLE 1. Individual Correlation between Different Cytokines and Clinical/ Radiographic Parameters Parameter IL-1b IL-6 IL-10 PGE2 TNF-a

SRL

EX

POP

TTP-POP

0.0742 P = .1934 0.6354 P = .0499* 0.3894 P = .2182 0.1946 P = .5305 0.0897 P = .7712

0.3759 P = .2338 0.2673 P = .3916 0.3678 P = .2435 0.4795 P = .1344 0.6394 P = .0491*

0.0447 P = .1628 0.8235 P = .0158* 0.9773 P = .0056* 0.2726 P = .3824 0.7248 P = .0302*

0.1074 P = .7281 0.6687 P = .0476* 0.6664 P = .0488* 0.1691 P = .5851 0.6488 P = .0490*

EX, exudation. *P < .05.

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TABLE 2. Correlations between Different Cytokines Considering the SRL as a Dependent Variable Correlation SRL IL-1b IL-1b IL-6 IL-10 PGE2 TNF-a

– – – – –

IL-6

IL-10

PGE2

TNF-a

0.5861* 0.3146 +0.2171 0.0572 – +0.0752 0.5749* 0.3574 – – 0.4466 0.2609 – – – +0.1612 – – – –

*P < .05.

exudation in root canals was correlated with higher levels of TNF-a, which is in accordance with previous investigations (20, 22, 23). Although positive (+) and negative (-) correlations were found between clinical/radiographic features and individual values of different cytokines, namely, SRL/IL6 (+), exudation/TNF-a (+), TTP-POP with IL-6 (), IL-10 and with TNF-a (), and the concomitant release of other several cytokines (e.g. IL-1b, IL-6, IL-10, PGE2, and TNF-a) should be taken into account in the development of clinical symptomatology and bone destruction by either amplifying or reducing the immune response via networks (33). The relationship between these cytokines is complex and in many ways difficult to establish. For example, the relation between IL-6 and PGE2 has pro- and anti-inflammatory sides (34). In the present study, although IL-6 and PGE2 was positively correlated to each other in POP, they were negatively related in the SRL. It is worth to point out that IL-6 can either induce or repress PGE2 and vice versa according to the duration of the injury, the bacterial load, the stimulated signaling pathways, and the cytokines (9). PGE2 has been quantified in teeth with exudation (26) and in periapical tissue (27), being directly and indirectly implicated with most of the inflammatory and destructive changes in apical lesions (eg, vasodilatation) and increasing vascular permeability and collagen degradation (27). Particularly, when exudation was set as a dependent variable, PGE2 was positively related to TNF-a and IL-1b. Shimauchi et al (26) reported that PGE2 synergistically enhances the collagenase response by IL-1. When clinical parameters indicative of inflammation in periodontal ligament were evaluated, IL-6 production was found to be positively correlated with IL-1b/PGE2 in POP and IL-1b in TTP-POP, elucidating its relationship with other mediators involved in clinical symptomatology. Furthermore, the ability of IL-6 to induce osteoclast formation and bone resorption (7, 8) was observed in its positive correlation with SRL as shown in Table 1. Particularly, when SRL was set as a dependent variable, IL-6 was negatively correlated with IL-1b/PGE2 production. These data may be explained by the chronic characteristic of apical periodontitis because bone resorption has a low progression rate and cytokine levels may not be so high. The role of IL-6 in human resorptive diseases such as periodontitis (35) and rheumatoid arthritis (36) has been reported. TABLE 3. Correlations between Different Cytokines Considering the Exudation (EX) as the Dependent Variable Correlation EX IL1b IL1b IL-6 IL-10 PGE2 TNF-a

– – – – –

IL-6

IL-10

PGE2

TNF-a

+0.4179 – – – –

+0.4250 +0.1255 – – –

+0.6335* 0.1152 +0.0143 – –

0.4611 +0.6815* +0.7656* +0.6599* –

*P < .05.

Clinical/Radiographic Features and Inflammatory Cytokines

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Clinical Research TABLE 4. Correlations between Different Cytokines Considering the POP as the Dependent Variable Correlation POP IL-1b IL-1b IL-6 IL-10 PGE2 TNF-a

– – – – –

IL-6

IL-10

PGE2

TNF-a

+0.9988† +0.9703† 0.0672 +0.8397* – +0.2588 +0.7576* +0.0434 – – +0.9883† +0.2956 – – – -0.6041* – – – –

*P < .05. † P < .01.

TABLE 5. Correlations between Different Cytokines Considering the TTP-POP as the Dependent Variable Correlation TTP-POP IL-1b – – – – –

*P < .05.

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Acknowledgments The authors deny any conflicts of interest related to this study.

References

IL-10 was included as an anti-inflammatory cytokine that counterbalances the production of proinflammatory cytokines, such as IL-1b, TNF-a, and PGE2 (37). Therefore, IL-10 also reduces the antigen presentation to immune cells in inflammatory sites. As expected, IL-10 was found to be negatively correlated with the presence of clinical symptomatology in POP and TTP-POP as shown in Table 1 (P < .05). When POP and TTP-POP were set as dependent variables, IL-10 levels increased in response to IL-1b and PGE2. Thus, particularly in teeth with exudation, IL-10 was positively related to the production of TNF-a. In the present study, these balances between anti- and proinflammatory cytokines characterize the chronic inflammatory form of the endodontic disease as teeth with pulp necrosis showed apical periodontitis. Considering POP as a dependent variable, IL-1b was present and positively related to increased levels of TNF-a production. Previously, Stashenko et al (38) showed that the production of these 2 mediators is coordinated in the periodontal tissues. Meanwhile, IL-1b was positively related to increased levels of IL-6 and PGE2. Interestingly, such findings suggest that TNF-a and IL-1b, which are pleiotropic inflammatory mediators, might play a role in initiation and up-regulation of the inflammatory response in apical periodontitis in addition to stimulating the production of secondary mediators such as IL-6 and PGE2. Research works involving the ccomprehension of apical periodontitis mechanisms and the developing of clinical and radiographic signs have emerged in the endodontic literature (39–44). Morsani et al (41) suggested that the specific genetic markers associated with increased IL-1b production might contribute to apical periodontitis increased susceptibility. Particularities regarding bacteria including virulence factors such as Porphyromonas gingivalis–FimA genotypes (39), and organization into biofilms (40) are also important factors related to the inflammatory process. Moreover, Siqueira et al (44) reported that polymorphism of Fc-gamma receptor genes might influence the patient’s response to endodontic treatment of teeth with apical periodontitis. Overall, the high reactivity of macrophage cells to bacteria and their by-products (eg, LPS) seems to be a susceptibility factor for the initiation of periapical tissue breakdown in endodontic diseases. Also, the selected cytokines involved in apical periodontitis have distinct

IL-1b IL-6 IL-10 PGE2 TNF-a

patterns of relationship in the clinical symptomatology. Because proand anti-inflammatory cytokines work in a network organization, further studies are necessary to elucidate the release patterns of other cytokines as well as to assess the clinical implications of these events on the development of clinical features.

Martinho et al.

IL-6

IL-10

PGE2

TNF-a

+0.8847* +0.5694* 0.1145 0.5734* – +0.1323 +0.5132 0.0215 – – +0.6890* +0.1115 – – – 0.4512 – – – –

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Clinical Research 26. Shimauchi H, Takayama S, Miki Y, Okada H. The change of periapical exudate prostaglandin E2 levels during root canal treatment. J Endod 1997;23:755–8. 27. McNicholas S, Torabinejad M, Blankenship J, Bakland L. The concentration of prostaglandin E2 in human periradicular lesions. J Endod 1991;17:97–100. 28. Barkhordar RA, Hayashi C, Hussain MZ. Detection of interleukin-6 in human dental pulp and periapical lesions. Endod Dent Traumatol 1999;15:26–7. 29. Martinho FC, Gomes BP. Quantification of endotoxins and cultivable bacteria in root canal infection before and after chemomechanical preparation with 2.5% sodium hypochlorite. J Endod 2008;34:268–72. 30. Martinho FC, Chiesa WM, Leite FR, Cirelli JA, Gomes BP. Antigenic activity of bacterial endodontic contents from primary root canal infection with periapical lesions against macrophage in the release of interleukin-1beta and tumor necrosis factor alpha. J Endod 2010;36:1766–9. 31. Jacinto RC, Gomes BP, Shah HN, Ferraz CC, Zaia AA, Souza-Filho FJ. Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth. J Med Microbiol 2005;54:777–83. 32. Jiang J, Zuo J, Hurst IR, Holliday LS. The synergistic effect of peptidoglycan and lipopolysaccaride on osteoclast formation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:738–43. 33. Stashenko P, Teles R, D’Souza R. Periapical inflammatory responses and their modulation. Crit Rev Oral Biol Med 1998;9:498–521. 34. Patil C, Rossa C, Kirkwood KL. Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin-6 expression through multiple mitogen-activated protein kinase pathways in periodontal ligament fibroblasts. Oral Microbiol Immunol 2006;21:392–8.

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35. Kono Y, Beagley KW, Fujihashi K, et al. Cytokine regulation of localized inflammation; induction of activated B cells and IL-6-mediated polyclonal IgG an IgA synthesis in inflamed human gingiva. J Immunol 1991;146:1812–21. 36. Al-Balaghi S, Strom H, M€oller E. B cell differentiation factor in synovial fluid of patients with rheumatoid arthritis. Immunol Rev 1984;78:7–23. 37. Correa FO, Gonc¸alves D, Figueredo CM, Bastos AS, Gustafsson A, Orrico SR. Effect of periodontal treatment on metabolic control, systemic inflammation and cytokines in patients with type 2 diabetes. J Clin Periodontol 2010;37:53–8. 38. Stashenko P, Jandinski JJ, Fujiyoshi P, Rynar J, Socransky SS. Tissue levels of bone resorptive cytokines in periodontal diseases. J Periodontol 1991;62:504–9. 39. Wang Q, Zhou XD, Zheng QH, Wang Y, Tang L, Huang DM. Distribution of Porphyromonas gingivalis fimA genotypes in chronic apical periodontitis associated with symptoms. J Endod 2010;36:1790–5. 40. Ricucci D, Siqueira JF Jr. Biofilms and apical periodontitis: study of prevalence and association with clinical and histopathologic findings. J Endod 2010;36: 1277–88. 41. Morsani JM, Aminoshariae A, Han YW, Montagnese TA, Mickel A. Genetic predisposition to persistent apical periodontitis. J Endod 2011;37:455–9. 42. Henriques LC, de Brito LC, Tavares WL, Vieira LQ, Ribeiro Sobrinho AP. Cytokine analysis in lesions refractory to endodontic treatment. J Endod 2011;37:1659–62. 43. Burgener B, Ford AR, Situ H, et al. Biologic markers for odontogenic periradicular periodontitis. J Endod 2010;36:1307–10. 44. Siqueira JF Jr, Rocas IN, Provenzano JC, Daibert FK, Silva MG, Lima KC. Relationship between Fcgamma receptor and interleukin-1 gene polymorphisms and posttreatment apical periodontitis. J Endod 2009;35:1186–92.

Clinical/Radiographic Features and Inflammatory Cytokines

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