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 corre- lation between different clinical signs/symptoms and radiographic features according to the levels of inter- leukin (IL)-1b, tumor necrosis factor a (TNF-a), IL-6, IL-10, prostaglandin E 2 (PGE 2 ), and their networks produced by endodontic content–stimulated macro- phages 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 macro- phages were stimulated with bacterial contents during 24 hs. The amounts of IL-1b, TNF-a, IL-6, IL-10 and PGE 2 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 PGE 2 , IL-1b, and TNF-a (P < .05), whereas IL-6 and PGE 2 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 pe- riodontitis, 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 E 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 E 2 (PGE 2 ), chemo- and cytokines, cellular adhesion molecules, and bone resorption–related factors (5, 6). IL-6 acts as a proinflammatory cytokine during periodontitis and stimulates osteo- clastic differentiation and bone resorption in chronic inflammatory periodontitis (7, 8). PGE 2 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). Lipopolysac- charides (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, PGE 2 , 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 Diag- nosis 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: bpgomes@fop.unicamp.br 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.02.021 Clinical Research 740 Martinho et al. JOE — Volume 38, Number 6, June 2012