Clinical Research
Immunoexpression of Transforming Growth Factor Beta and Interferon Gamma in Radicular and Dentigerous Cysts Maiara de Moraes, DDS, PhD,* Pedro Carlos da Rocha Neto, DDS, MSc,* Felipe Rodrigues de Matos, DDS, PhD,* Maria Luiza Diniz de Sousa Lopes, DDS, MSc,* Paulo Roberto Medeiros de Azevedo, MSc,† and Antonio de Lisboa Lopes Costa, DDS, PhD* Abstract Introduction: The aim of this study was to evaluate and compare the immunohistochemical expression of transforming growing factor beta (TGF-b) and interferon gamma (IFN-g) between radicular cysts (RCs) and dentigerous cysts (DCs). Methods: Twenty RCs and DCs were selected for analysis of the immunoexpression of TGF-b and IFN-g in the epithelium and capsule. Results: The cell reactivity of TGF-b and IFN-g in the lining epithelium and capsule of RCs showed no significant differences when compared with DCs (P > .05). There was a tendency of a higher expression of TGF-b in the capsule of DCs. Conclusions: Our results showed the presence of TGF-b and IFN-g in RCs and DCs, supporting the hypothesis that both participate in the development of these lesions, where IFN-g usually plays a role in bone resorption, which is counterbalanced by the osteoprotective activity performed by TGF-b. (J Endod 2014;40:1293–1297)
Key Words Dentigerous cyst, immunohistochemistry, interferon gamma, radicular cyst, transforming growth factors
From the *Postgraduate Program, Oral Pathology and Department of Statistics, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil. Address requests for reprints to Dr Antonio de Lisboa Lopes Costa, Universidade Federal do Rio Grande do Norte, Departamento de Odontologia, Av Senador Salgado Filho, 1787 Lagoa Nova, Natal, RN, Brazil CEP 59056-000. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.010 †
O
dontogenic cysts are one of the most common osseous-destructive lesions in the jaws. Traditionally, they are classified as developmental cysts, including dentigerous cysts (DCs), and inflammatory cysts, such as radicular cysts (RCs). The origin of inflammatory cysts is thought to be from an inflammatory process, whereas the origin of developmental cysts remains unknown; however, it does not seem to be related to an inflammatory process (1–3). The precise biological mechanisms of the development and enlargement of jaw cysts have not been completely enlightened. Disturbances in the number and activity of osteoclastic cells cause most of the bone diseases, resulting in improper bone resorption, which exceeds the compensatory capacity of osteoblasts (1, 4). The regulation of osteoclast biology and bone metabolism implies various mediators, including receptor activator of nuclear factor kappa-B (RANK), receptor activator of nuclear factor kappaB ligand (RANKL), and osteoprotegerin (OPG), (1, 5, 6) and their complex interrelationships with cytokines and growth factors, such as interferon-gamma (IFN-g) and transforming growth factor-beta (TGF-b), respectively (7–9). IFN-g has been implicated as an inductor of osteoclastogenesis by inducing the synthesis of RANKL (9–11). On the other hand, IFN-g may suppress osteoclast differentiation induced by RANKL, inhibiting bone resorption (12, 13). Therefore, the final effect of IFN-g on bone tissue depends on the imbalance between its osteoprotective and osteoresorption activity, which under circumstances of inflammatory and infectious diseases, is in favor of bone resorption (9, 11). TGF-b has also been associated with bone resorption, representing an indispensable factor in RANKL-induced osteoclastogenesis (7, 14). However, TGF-b may act as a stimulator of bone formation through a favorable effect in osteoblastogenesis (15–17). Recent studies have shown the participation of IFN-g and TGF-b in inflammatory periapical lesions (8, 10, 18–22). The purpose of this study was to evaluate and compare the immunohistochemical expression of IFN-g and TGF-b between DCs and RCs.
Materials and Methods The original hematoxylin-eosin–stained slides and formalin-fixed paraffinembedded specimen blocks of all RCs and DCs diagnosed between January 2000 and October 2009 were retrieved from the files of the Oral Pathology Service at Federal University of Rio Grande do Norte (UFRN), Natal, Brazil, from the Department of Histopathology. The hematoxylin-eosin slides were reviewed to confirm the diagnosis. The tissues were classified as cysts whenever a partial or total epithelium lining was present. The diagnosis of cysts was based mainly on radiographic and histopathologic examination. DCs showing intense inflammation and cysts with inadequate tissue samples were excluded. A total of 20 RCs and 20 DCs were selected for the study. The clinical and radiographic information, including age, sex, and anatomic site, were obtained from biopsy forms submitted by the clinicians.
Immunohistochemistry For the immunohistochemical reactions, the tissue section samples were deparaffinized with xylene, rehydrated in graded alcohols, and washed in deionized water and phosphate-buffered saline (PBS). Samples were then incubated with 3% hydrogen
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Cell Counting The immunoexpression of TGF-b and INF-g was evaluated in lining epithelium and fibrous capsule. The epithelial immunoexpression was quantitatively evaluated by 2 observers using 400 magnification and classified according to the following scores: 0: no staining (76%). In the fibrous capsule, the analysis was quantitative, and the number of positive cells was counted in 10 representative and consecutive microscopic highpower fields (1000) over totally counted cells irrespective of the cell type. Digital images were loaded on the software IMAGE J (National Institutes of Health, Bethesda, MD) to count the number of immunostained cells. The results are expressed as the mean percentage of observations per field, with the following modifications. Based on this mean percentage, a score was determined for each case, taking into account the standard scoring system used for the lining epithelium: 0: no staining (75% positive cells) in RCs (75%) (Fig. 2) and DCs (85%) (Fig. 3), with no significant differences between the groups (P = .620). INF-g exhibited positivity in the basal and suprabasal epithelial cells in RCs (Fig. 1C) and DCs (Fig. 1D). The analyses of the immunoreactivity of INF-g according to the percentage of the scores revealed a predominance of score 3 (>75% positive cells) in RCs (85%) (Fig. 2) and DCs (75%) (Fig. 3), with no significant differences between groups (P = .565).
Quantitative Analysis of Fibrous Capsule With regard to reactivity for TGF-b and INF-g in the stromal cells, the presence of positive fibroblasts, polymorphonuclear neutrophils, plasmocytes, lymphocytes, and macrophage cells were observed. The immunoreactivity was predominantly in the cytoplasm. Figure 3 summarizes the quantitative analysis of lesions immunostained for TGF-b and INF-g in the fibrous capsule. TGF-b revealed a predominance of score 3 (>75% positive cells) in RCs (80%) (Fig. 2) and DCs (95%) (Fig. 3), with no significant differences between groups (P = .429). The analyses of the immunoreactivity of INF-g according to the percentage of the scores revealed a predominance of score 3 (>75% positive cells) in RCs (75%) (Fig. 2) and DCs (60%) (Fig. 3), with no significant differences between groups (P = .429).
Discussion Cyst formation is believed to be related to the release of cytokines and growth factors leading to the activation of the proliferation of epithelial remnants (23). In the present study, we have examined the immunoexpression to IFN-g and TGF-b in RCs and DCs. Although presenting as pathological entities of a different nature, the presence of these molecules may act in a direct or indirect way favoring the enlargement of the cysts. Irrespective of the stimulus for the development of injury, the presence of a Th1 or Th2 immune response may lead to changes in TGF-b and IFN-gamma immunoexpression through costimulation with proinflammatory cytokines. For RCs, the presence of inflammation promotes the release of proresorptive factors. In DC development, there may be indirect stimuli associated with the activation of other factors such as the vascular endothelial growth factor (VEGF), which may act as substitute for macrophage colony-stimulating factor (M-CSF) in the support of osteoclastic bone resorption (1, 24). Furthermore, as suggested by our team in previous studies (1, 24), an increased expression of factors such as RANKL and VEGF may induce the differentiation and survival of osteoclasts. In this way, by the frequent presence of hemorrhagic areas in the capsule of DCs, we can assume that the increased immunoreactivity of VEGF in this entity may contribute to bone resorption and cystic expansion because VEGF acts as an important molecule for the recruitment of osteoclasts. The immunoinflammatory reaction in periapical lesions causes the activation of a Th1 immune response was characterized by the production of proinflammatory cytokines such as IFN-g, tumor necrosis factor alpha (TNF-a), and interleukin (IL)-1, IL-2, and IL-12, and the Th2 response was characterized by synthesis of anti-inflammatory cytokines such as IL-4, IL-5, IL-6, IL-10, and IL-13. In addition to these cytokines, there is also the presence of growth factors such as TGF-b. Th1 and Th2 have opposite activities; in the context of periapical lesions, it is believed that a Th1 immune response with its major cytokine IFN-g is associated with progression and the process of bone resorption, whereas the immunosuppressive action of TGF-b and Th2 cytokines acts in the modulation of the immune-inflammatory response and the repair of damaged bone tissue (8, 9, 25–28). JOE — Volume 40, Number 9, September 2014
Clinical Research
Figure 1. (A) An RC with strong TGF-b expression in lining epithelium (arrow) and fibrous capsule (arrowhead). (B) A DC with strong TGF-b expression in lining epithelium (arrow) and fibrous capsule (arrowhead). (C) An RC with weak INF-g expression in lining epithelium (arrow) and fibrous capsule (arrowhead). (D) A DC with strong INF-g expression in lining epithelium (arrow) and fibrous capsule (arrowhead). ADVANCE HRP method, original magnifications: 400.
In this context, it has been proposed that IFN-g plays a role in osseous metabolism as a cytokine that stimulates bone resorption in certain inflammatory diseases. Kotake et al (11) cultivated T-cell IFN-g+ along with monocyte precursors of osteoclasts from an inflammatory disease (rheumatoid arthritis) and observed osteoclast differentiation from monocytes as well as an increased expression of RANKL. They conclude that T cells producing IFN-g induced osteoclastogenesis through the expression of RANKL. Another experiment tested if IFN-g positively modulates osteoclastogenesis mediated by RANKL during the progression of periodontal disease induced by Actinobacillus actinomycetemcomitans in mice and humans (10). The simultaneous expression of IFN-g and RANKL in T cells reactive to the microorganism in all experiments confirmed this hypothesis. Despite the vast amount of studies showing the IFN-g as a boneresorptive stimulator, in other studies this cytokine has been linked as
an inhibitor of bone resorption through an antiosteoclastogenesis effect (13, 29–31). The in vivo experiments of Gao et al (9) highlight the contradictory actions of IFN-g on bone tissue, whereas this cytokine has both the potential to inhibit osteoclast differentiation by acting directly on osteoclast precursor cells or to indirectly promote osteoclastogenesis, inducting the major histocompatibility complex class II antigen presentation by macrophages with subsequent activation of T lymphocytes to produce osteoclastogenic factors like RANKL and TNF-a. These authors concluded that the effect of IFN-g in bone tissue is because of the imbalance between their osteoprotector and resorption activity, which under inflammatory conditions and infectious diseases is in favor of bone resorption. Therefore, it can be inferred that RANKL produced in these studies would be responsible for osteoclastic differentiation observed and no IFN-g by itself. When the RANKL induces terminal differentiation of osteoclast precursor cells, they become resistant to the
Figure 2. Percentages of the scores of TGF-b–and INF-g–positive cells in an RC. Score 0 or no staining (76%).
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Figure 3. Percentages of the scores of TGF-b–and INF-g–positive cells in a DC. Score 0 or no staining (76%).
inhibitory effect of IFN-g (31). Furthermore, it is known that IFN-g is a potent stimulator of macrophage secretion of IL-1 and TNF-a, 2 cytokines related to periapical bone destruction (27). IL-1 has been linked to the inflammatory response and the stimulation of proliferation of the epithelial cell rests of Malassez involved in RC development (23). In this light, the presence of IFN-g in odontogenic cysts suggests an osteoreabsorptive action of this cytokine, which is vital to cystic growth. The immunoexpression of IFN-g between RCs and DCs in the epithelia or capsule showed no significant difference. The expression of IFN-g in RCs is well documented although there is no study reporting its expression in DCs. However, the comparison of IFN-g expression between RCs and periapical granulomas (PGs) has been investigated revealing a similar (18) and higher (8) expression of IFN-g in RCs when compared with PGs. Ihan Hren and Ihan (19) compared the inflammatory response of RCs, PGs streptococcus, and PGs anaerobic bacteria and found that the production of IFN-g is higher in RCs than in the PG streptococcus group but is similar to PGs anaerobic bacteria. de Carvalho Fraga et al (32) found higher levels of IFN- g and lower IL-4 (Th2-like cytokine) levels in the RCs than in the PGs in their study. Even though the signaling pathways mediated by these cytokines are unclear, these authors emphasize that it is likely that the RC environment is mainly influenced by the Th1 immune response, whereas a Th2 response would be more related to chronic lesions. Moreover, Campos et al (33) recently reported that epigenetic regulation, through partial or total methylation of IFN-g gene transcription, might be involved in the modulation of IFN-g secretion in periapical lesions. These authors found an increase in the methylation profile in RCs compared with PGs although they could not make conclusions about the potential role of methylation in RC development because the causes or consequences of this alteration are not clear and may depend on variables such as bacterial infection. Controversial results have been obtained about the actions of TGFb on bone metabolism. Many authors have attributed to this growth factor an osteoblastogenesis effect (15–17, 34), but others have associated it with osteoclastogenesis (7, 14, 35, 36). Tyler et al (22) demonstrated TGF-b1 mRNA and protein in periapical cysts and granulomas and assigns it a role in the repair process. Piattelli et al (20) observed a higher expression of TGF-b1 in odontogenic keratocysts than in RCs and DCs and associated this fact to the higher growth potential of keratocysts by intense cell proliferation, which would be regulated by TGF-b1. Gazivoda et al (27) added IL-10 and TGF-b to inflammatory periapical lesion cell cultures and evaluated the synthesis of proinflamma1296
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tory cytokines. They showed that both IL-10 and TGF-b were able to inhibit the production of IL-1, TNF-a, and IL-8 (bone resorption factors), with TGF-b showing the higher immunosuppressive activity. However, despite these conflicting findings, a high expression of TGF-b1 during healing of bone fractures has been observed, suggesting its involvement in the bone repair process (37–39). In a literature review, Janssens et al (38) analyzed 39 studies that investigated the effect of TGF-b1 on bone tissue, and they concluded that TGF-b1 promotes the healing of bone fractures and bone formation in vivo. They reported that TGF-b1 is a growth factor that can be considered for use as an osteogenic agent in bone fractures or as an adjunct in excessive bone resorption observed in osteoporosis. Furthermore, TGF-b1 regulates a great number of biological processes such as proliferation, survival, differentiation, recognition, apoptosis, cell migration, and production of extracellular matrix, thus having important functions in immune responses, angiogenesis, wound healing, and bone metabolism. The tendency toward a greater expression of TGFb in the fibrous capsule of DCs found in this study may be related to increased angiogenesis, which was already discussed in a previous study (24). Two studies using primary bone marrow cell cultures (14, 40) and another study using murine monocytes (41) showed the induction of osteoclastogenesis by TGF-b. In the last study, the authors observed high levels of RANKL, M-CSF, and TGF-b, which were associated with an increase of osteoclast development from preosteoclast cells/monocytes shown by the presence of tartrate-resistant acid phosphatase in multinucleated cells and the expression of other osteoclast-specific markers such as calcitonin receptor and vitronectin receptor. According to these authors, TGF-b acts on preosteoclasts, promoting the differentiation of these cells into osteoclasts. In the present study, no significant difference was observed in the immunoexpression of TGF-b between RCs and DCs in the epithelia or capsule. The presence of TGF-b1 in odontogenic cysts assumes a compensatory mechanism by which the inflammatory response acts in the repair and remodeling of bone-injured tissue (8, 22). We found no statistically significant difference of TGF-b and IFN-g in RCs and DCs and believe that the balance of these proteins observed in immunostaining may be favor an osteolytic activity as well as an osteogenic activity in the odontogenic cysts. These antagonistic actions may depend on the secretion of other proteins such as RANKL, VEGF, matrix metalloproteinases (MMPs), and proinflammatory cytokines such as IL-1 and TNF-a that should be analyzed together for a more effective JOE — Volume 40, Number 9, September 2014
Clinical Research conclusion about the involvement of these proteins in bone resorption and cystic expansion of the odontogenic cysts.
Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. de Moraes M, de Lucena HF, de Azevedo PR, et al. Comparative immunohistochemical expression of RANK, RANKL and OPG in radicular and dentigerous cysts. Arch Oral Biol 2011;56:1256–63. 2. de Souza LB, Gordon-Nunez MA, Nonaka CF, et al. Odontogenic cysts: demographic profile in a Brazilian population over a 38-year period. Med Oral Patol Oral Cir Bucal 2010;15:e583–90. 3. Avelar RL, Antunes AA, Carvalho RW, et al. Odontogenic cysts: a clinicopathological study of 507 cases. J Oral Sci 2009;51:581–6. 4. Aeschlimann D, Evans BA. The vital osteoclast: how is it regulated? Cell Death Differ 2004;11(suppl 1):S5–7. 5. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337–42. 6. Menezes R, Bramante CM, da Silva Paiva KB, et al. Receptor activator NFkappaBligand and osteoprotegerin protein expression in human periapical cysts and granulomas. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:404–9. 7. Yasui T, Kadono Y, Nakamura M, et al. Regulation of RANKL-induced osteoclastogenesis by TGF-beta through molecular interaction between Smad3 and Traf6. J Bone Miner Res 2011;26:1447–56. 8. Teixeira-Salum TB, Rodrigues DB, Gervasio AM, et al. Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts. J Oral Pathol Med 2010;39:250–6. 9. Gao Y, Grassi F, Ryan MR, et al. IFN-gamma stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J Clin Invest 2007;117:122–32. 10. Teng YT, Mahamed D, Singh B. Gamma interferon positively modulates Actinobacillus actinomycetemcomitans-specific RANKL+ CD4+ Th-cell-mediated alveolar bone destruction in vivo. Infect Immun 2005;73:3453–61. 11. Kotake S, Nanke Y, Mogi M, et al. IFN-gamma-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL. Eur J Immunol 2005;35:3353–63. 12. Queiroz-Junior CM, Silva MJ, Correa JD, et al. A controversial role for IL-12 in immune response and bone resorption at apical periodontal sites. Clin Dev Immunol 2010;2010:327417. 13. Ji JD, Park-Min KH, Shen Z, et al. Inhibition of RANK expression and osteoclastogenesis by TLRs and IFN-gamma in human osteoclast precursors. J Immunol 2009;183:7223–33. 14. Fuller K, Lean JM, Bayley KE, et al. A role for TGFbeta(1) in osteoclast differentiation and survival. J Cell Sci 2000;113(Pt 13):2445–53. 15. Tachi K, Takami M, Sato H, et al. Enhancement of bone morphogenetic protein-2induced ectopic bone formation by transforming growth factor-beta1. Tissue Eng Part A 2011;17:597–606. 16. Zhou S. TGF-beta regulates beta-catenin signaling and osteoblast differentiation in human mesenchymal stem cells. J Cell Biochem 2011;112:1651–60. 17. Smith EL, Colombo JS, Sloan AJ, et al. TGF-beta1 exposure from bone surfaces by chemical treatment modalities. Eur Cell Mater 2011;21:193–201. 18. Fukada SY, Silva TA, Garlet GP, et al. Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases. Oral Microbiol Immunol 2009;24:25–31. 19. Ihan Hren N, Ihan A. T lymphocyte activation and cytokine expression in periapical granulomas and radicular cysts. Arch Oral Biol 2009;54:156–61.
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20. Piattelli A, Rubini C, Fioroni M, et al. Expression of transforming growth factor-beta 1 (TGF-beta 1) in odontogenic cysts. Int Endod J 2004;37:7–11. 21. Danin J, Linder LE, Lundqvist G, et al. Tumor necrosis factor-alpha and transforming growth factor-beta1 in chronic periapical lesions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;90:514–7. 22. Tyler LW, Matossian K, Todd R, et al. Eosinophil-derived transforming growth factors (TGF-alpha and TGF-beta 1) in human periradicular lesions. J Endod 1999;25: 619–24. 23. Keinan D, Cohen RE. The significance of epithelial rests of Malassez in the periodontal ligament. J Endod 2013;39:582–7. 24. de Moraes M, de Matos FR, de Souza LB, et al. Immunoexpression of RANK, RANKL, OPG, VEGF, and vWF in radicular and dentigerous cysts. J Oral Pathol Med 2013;42: 468–73. 25. Stashenko P, Goncalves RB, Lipkin B, et al. Th1 immune response promotes severe bone resorption caused by Porphyromonas gingivalis. Am J Pathol 2007;170: 203–13. 26. Colic M, Lukic A, Vucevic D, et al. Correlation between phenotypic characteristics of mononuclear cells isolated from human periapical lesions and their in vitro production of Th1 and Th2 cytokines. Arch Oral Biol 2006;51:1120–30. 27. Gazivoda D, Dzopalic T, Bozic B, et al. Production of proinflammatory and immunoregulatory cytokines by inflammatory cells from periapical lesions in culture. J Oral Pathol Med 2009;38:605–11. 28. Walker KF, Lappin DF, Takahashi K, et al. Cytokine expression in periapical granulation tissue as assessed by immunohistochemistry. Eur J Oral Sci 2000;108: 195–201. 29. Takayanagi H, Kim S, Taniguchi T. Signaling crosstalk between RANKL and interferons in osteoclast differentiation. Arthritis Res 2002;4(suppl 3):S227–32. 30. Mermut S, Bengi AO, Akin E, et al. Effects of interferon-gamma on bone remodeling during experimental tooth movement. Angle Orthod 2007;77:135–41. 31. Huang W, O’Keefe RJ, Schwarz EM. Exposure to receptor-activator of NFkappaB ligand renders pre-osteoclasts resistant to IFN-gamma by inducing terminal differentiation. Arthritis Res Ther 2003;5:R49–59. 32. de Carvalho Fraga CA, Alves LR, de Sousa AA, et al. Th1 and Th2-like protein balance in human inflammatory radicular cysts and periapical granulomas. J Endod 2013; 39:453–5. 33. Campos K, Gomes CC, de Fatima Correia-Silva J, et al. Methylation pattern of IFNG in periapical granulomas and radicular cysts. J Endod 2013;39:493–6. 34. Ripamonti U, Roden LC. Induction of bone formation by transforming growth factorbeta2 in the non-human primate Papio ursinus and its modulation by skeletal muscle responding stem cells. Cell Prolif 2010;43:207–18. 35. Kawakatsu M, Kanno S, Gui T, et al. Loss of Smad3 gives rise to poor soft callus formation and accelerates early fracture healing. Exp Mol Pathol 2011;90: 107–15. 36. McGowan NW, MacPherson H, Janssens K, et al. A mutation affecting the latencyassociated peptide of TGFbeta1 in Camurati-Engelmann disease enhances osteoclast formation in vitro. J Clin Endocrinol Metab 2003;88:3321–6. 37. Sarahrudi K, Thomas A, Mousavi M, et al. Elevated transforming growth factor-beta 1 (TGF-beta1) levels in human fracture healing. Injury 2011;42:833–7. 38. Janssens K, ten Dijke P, Janssens S, et al. Transforming growth factor-beta1 to the bone. Endocr Rev 2005;26:743–74. 39. Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res 2002;17:513–20. 40. Yamaguchi M, Kishi S. Differential effects of transforming growth factor-beta on osteoclast-like cell formation in mouse marrow culture: relation to the effect of zinc-chelating dipeptides. Peptides 1995;16:1483–8. 41. Yan T, Riggs BL, Boyle WJ, et al. Regulation of osteoclastogenesis and RANK expression by TGF-beta1. J Cell Biochem 2001;83:320–5.
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Immunoexpression of Transforming Growth Factor Beta and Interferon Gamma in Radicular and Dentigerous Cysts Maiara de Moraes, DDS, PhD,* Pedro Carlos da Rocha Neto, DDS, MSc,* Felipe Rodrigues de Matos, DDS, PhD,* Maria Luiza Diniz de Sousa Lopes, DDS, MSc,* Paulo Roberto Medeiros de Azevedo, MSc,† and Antonio de Lisboa Lopes Costa, DDS, PhD* Abstract Introduction: The aim of this study was to evaluate and compare the immunohistochemical expression of transforming growing factor beta (TGF-b) and interferon gamma (IFN-g) between radicular cysts (RCs) and dentigerous cysts (DCs). Methods: Twenty RCs and DCs were selected for analysis of the immunoexpression of TGF-b and IFN-g in the epithelium and capsule. Results: The cell reactivity of TGF-b and IFN-g in the lining epithelium and capsule of RCs showed no significant differences when compared with DCs (P > .05). There was a tendency of a higher expression of TGF-b in the capsule of DCs. Conclusions: Our results showed the presence of TGF-b and IFN-g in RCs and DCs, supporting the hypothesis that both participate in the development of these lesions, where IFN-g usually plays a role in bone resorption, which is counterbalanced by the osteoprotective activity performed by TGF-b. (J Endod 2014;40:1293–1297)
Key Words Dentigerous cyst, immunohistochemistry, interferon gamma, radicular cyst, transforming growth factors
From the *Postgraduate Program, Oral Pathology and Department of Statistics, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil. Address requests for reprints to Dr Antonio de Lisboa Lopes Costa, Universidade Federal do Rio Grande do Norte, Departamento de Odontologia, Av Senador Salgado Filho, 1787 Lagoa Nova, Natal, RN, Brazil CEP 59056-000. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.010 †
O
dontogenic cysts are one of the most common osseous-destructive lesions in the jaws. Traditionally, they are classified as developmental cysts, including dentigerous cysts (DCs), and inflammatory cysts, such as radicular cysts (RCs). The origin of inflammatory cysts is thought to be from an inflammatory process, whereas the origin of developmental cysts remains unknown; however, it does not seem to be related to an inflammatory process (1–3). The precise biological mechanisms of the development and enlargement of jaw cysts have not been completely enlightened. Disturbances in the number and activity of osteoclastic cells cause most of the bone diseases, resulting in improper bone resorption, which exceeds the compensatory capacity of osteoblasts (1, 4). The regulation of osteoclast biology and bone metabolism implies various mediators, including receptor activator of nuclear factor kappa-B (RANK), receptor activator of nuclear factor kappaB ligand (RANKL), and osteoprotegerin (OPG), (1, 5, 6) and their complex interrelationships with cytokines and growth factors, such as interferon-gamma (IFN-g) and transforming growth factor-beta (TGF-b), respectively (7–9). IFN-g has been implicated as an inductor of osteoclastogenesis by inducing the synthesis of RANKL (9–11). On the other hand, IFN-g may suppress osteoclast differentiation induced by RANKL, inhibiting bone resorption (12, 13). Therefore, the final effect of IFN-g on bone tissue depends on the imbalance between its osteoprotective and osteoresorption activity, which under circumstances of inflammatory and infectious diseases, is in favor of bone resorption (9, 11). TGF-b has also been associated with bone resorption, representing an indispensable factor in RANKL-induced osteoclastogenesis (7, 14). However, TGF-b may act as a stimulator of bone formation through a favorable effect in osteoblastogenesis (15–17). Recent studies have shown the participation of IFN-g and TGF-b in inflammatory periapical lesions (8, 10, 18–22). The purpose of this study was to evaluate and compare the immunohistochemical expression of IFN-g and TGF-b between DCs and RCs.
Materials and Methods The original hematoxylin-eosin–stained slides and formalin-fixed paraffinembedded specimen blocks of all RCs and DCs diagnosed between January 2000 and October 2009 were retrieved from the files of the Oral Pathology Service at Federal University of Rio Grande do Norte (UFRN), Natal, Brazil, from the Department of Histopathology. The hematoxylin-eosin slides were reviewed to confirm the diagnosis. The tissues were classified as cysts whenever a partial or total epithelium lining was present. The diagnosis of cysts was based mainly on radiographic and histopathologic examination. DCs showing intense inflammation and cysts with inadequate tissue samples were excluded. A total of 20 RCs and 20 DCs were selected for the study. The clinical and radiographic information, including age, sex, and anatomic site, were obtained from biopsy forms submitted by the clinicians.
Immunohistochemistry For the immunohistochemical reactions, the tissue section samples were deparaffinized with xylene, rehydrated in graded alcohols, and washed in deionized water and phosphate-buffered saline (PBS). Samples were then incubated with 3% hydrogen
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Cell Counting The immunoexpression of TGF-b and INF-g was evaluated in lining epithelium and fibrous capsule. The epithelial immunoexpression was quantitatively evaluated by 2 observers using 400 magnification and classified according to the following scores: 0: no staining (76%). In the fibrous capsule, the analysis was quantitative, and the number of positive cells was counted in 10 representative and consecutive microscopic highpower fields (1000) over totally counted cells irrespective of the cell type. Digital images were loaded on the software IMAGE J (National Institutes of Health, Bethesda, MD) to count the number of immunostained cells. The results are expressed as the mean percentage of observations per field, with the following modifications. Based on this mean percentage, a score was determined for each case, taking into account the standard scoring system used for the lining epithelium: 0: no staining (75% positive cells) in RCs (75%) (Fig. 2) and DCs (85%) (Fig. 3), with no significant differences between the groups (P = .620). INF-g exhibited positivity in the basal and suprabasal epithelial cells in RCs (Fig. 1C) and DCs (Fig. 1D). The analyses of the immunoreactivity of INF-g according to the percentage of the scores revealed a predominance of score 3 (>75% positive cells) in RCs (85%) (Fig. 2) and DCs (75%) (Fig. 3), with no significant differences between groups (P = .565).
Quantitative Analysis of Fibrous Capsule With regard to reactivity for TGF-b and INF-g in the stromal cells, the presence of positive fibroblasts, polymorphonuclear neutrophils, plasmocytes, lymphocytes, and macrophage cells were observed. The immunoreactivity was predominantly in the cytoplasm. Figure 3 summarizes the quantitative analysis of lesions immunostained for TGF-b and INF-g in the fibrous capsule. TGF-b revealed a predominance of score 3 (>75% positive cells) in RCs (80%) (Fig. 2) and DCs (95%) (Fig. 3), with no significant differences between groups (P = .429). The analyses of the immunoreactivity of INF-g according to the percentage of the scores revealed a predominance of score 3 (>75% positive cells) in RCs (75%) (Fig. 2) and DCs (60%) (Fig. 3), with no significant differences between groups (P = .429).
Discussion Cyst formation is believed to be related to the release of cytokines and growth factors leading to the activation of the proliferation of epithelial remnants (23). In the present study, we have examined the immunoexpression to IFN-g and TGF-b in RCs and DCs. Although presenting as pathological entities of a different nature, the presence of these molecules may act in a direct or indirect way favoring the enlargement of the cysts. Irrespective of the stimulus for the development of injury, the presence of a Th1 or Th2 immune response may lead to changes in TGF-b and IFN-gamma immunoexpression through costimulation with proinflammatory cytokines. For RCs, the presence of inflammation promotes the release of proresorptive factors. In DC development, there may be indirect stimuli associated with the activation of other factors such as the vascular endothelial growth factor (VEGF), which may act as substitute for macrophage colony-stimulating factor (M-CSF) in the support of osteoclastic bone resorption (1, 24). Furthermore, as suggested by our team in previous studies (1, 24), an increased expression of factors such as RANKL and VEGF may induce the differentiation and survival of osteoclasts. In this way, by the frequent presence of hemorrhagic areas in the capsule of DCs, we can assume that the increased immunoreactivity of VEGF in this entity may contribute to bone resorption and cystic expansion because VEGF acts as an important molecule for the recruitment of osteoclasts. The immunoinflammatory reaction in periapical lesions causes the activation of a Th1 immune response was characterized by the production of proinflammatory cytokines such as IFN-g, tumor necrosis factor alpha (TNF-a), and interleukin (IL)-1, IL-2, and IL-12, and the Th2 response was characterized by synthesis of anti-inflammatory cytokines such as IL-4, IL-5, IL-6, IL-10, and IL-13. In addition to these cytokines, there is also the presence of growth factors such as TGF-b. Th1 and Th2 have opposite activities; in the context of periapical lesions, it is believed that a Th1 immune response with its major cytokine IFN-g is associated with progression and the process of bone resorption, whereas the immunosuppressive action of TGF-b and Th2 cytokines acts in the modulation of the immune-inflammatory response and the repair of damaged bone tissue (8, 9, 25–28). JOE — Volume 40, Number 9, September 2014
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Figure 1. (A) An RC with strong TGF-b expression in lining epithelium (arrow) and fibrous capsule (arrowhead). (B) A DC with strong TGF-b expression in lining epithelium (arrow) and fibrous capsule (arrowhead). (C) An RC with weak INF-g expression in lining epithelium (arrow) and fibrous capsule (arrowhead). (D) A DC with strong INF-g expression in lining epithelium (arrow) and fibrous capsule (arrowhead). ADVANCE HRP method, original magnifications: 400.
In this context, it has been proposed that IFN-g plays a role in osseous metabolism as a cytokine that stimulates bone resorption in certain inflammatory diseases. Kotake et al (11) cultivated T-cell IFN-g+ along with monocyte precursors of osteoclasts from an inflammatory disease (rheumatoid arthritis) and observed osteoclast differentiation from monocytes as well as an increased expression of RANKL. They conclude that T cells producing IFN-g induced osteoclastogenesis through the expression of RANKL. Another experiment tested if IFN-g positively modulates osteoclastogenesis mediated by RANKL during the progression of periodontal disease induced by Actinobacillus actinomycetemcomitans in mice and humans (10). The simultaneous expression of IFN-g and RANKL in T cells reactive to the microorganism in all experiments confirmed this hypothesis. Despite the vast amount of studies showing the IFN-g as a boneresorptive stimulator, in other studies this cytokine has been linked as
an inhibitor of bone resorption through an antiosteoclastogenesis effect (13, 29–31). The in vivo experiments of Gao et al (9) highlight the contradictory actions of IFN-g on bone tissue, whereas this cytokine has both the potential to inhibit osteoclast differentiation by acting directly on osteoclast precursor cells or to indirectly promote osteoclastogenesis, inducting the major histocompatibility complex class II antigen presentation by macrophages with subsequent activation of T lymphocytes to produce osteoclastogenic factors like RANKL and TNF-a. These authors concluded that the effect of IFN-g in bone tissue is because of the imbalance between their osteoprotector and resorption activity, which under inflammatory conditions and infectious diseases is in favor of bone resorption. Therefore, it can be inferred that RANKL produced in these studies would be responsible for osteoclastic differentiation observed and no IFN-g by itself. When the RANKL induces terminal differentiation of osteoclast precursor cells, they become resistant to the
Figure 2. Percentages of the scores of TGF-b–and INF-g–positive cells in an RC. Score 0 or no staining (76%).
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Figure 3. Percentages of the scores of TGF-b–and INF-g–positive cells in a DC. Score 0 or no staining (76%).
inhibitory effect of IFN-g (31). Furthermore, it is known that IFN-g is a potent stimulator of macrophage secretion of IL-1 and TNF-a, 2 cytokines related to periapical bone destruction (27). IL-1 has been linked to the inflammatory response and the stimulation of proliferation of the epithelial cell rests of Malassez involved in RC development (23). In this light, the presence of IFN-g in odontogenic cysts suggests an osteoreabsorptive action of this cytokine, which is vital to cystic growth. The immunoexpression of IFN-g between RCs and DCs in the epithelia or capsule showed no significant difference. The expression of IFN-g in RCs is well documented although there is no study reporting its expression in DCs. However, the comparison of IFN-g expression between RCs and periapical granulomas (PGs) has been investigated revealing a similar (18) and higher (8) expression of IFN-g in RCs when compared with PGs. Ihan Hren and Ihan (19) compared the inflammatory response of RCs, PGs streptococcus, and PGs anaerobic bacteria and found that the production of IFN-g is higher in RCs than in the PG streptococcus group but is similar to PGs anaerobic bacteria. de Carvalho Fraga et al (32) found higher levels of IFN- g and lower IL-4 (Th2-like cytokine) levels in the RCs than in the PGs in their study. Even though the signaling pathways mediated by these cytokines are unclear, these authors emphasize that it is likely that the RC environment is mainly influenced by the Th1 immune response, whereas a Th2 response would be more related to chronic lesions. Moreover, Campos et al (33) recently reported that epigenetic regulation, through partial or total methylation of IFN-g gene transcription, might be involved in the modulation of IFN-g secretion in periapical lesions. These authors found an increase in the methylation profile in RCs compared with PGs although they could not make conclusions about the potential role of methylation in RC development because the causes or consequences of this alteration are not clear and may depend on variables such as bacterial infection. Controversial results have been obtained about the actions of TGFb on bone metabolism. Many authors have attributed to this growth factor an osteoblastogenesis effect (15–17, 34), but others have associated it with osteoclastogenesis (7, 14, 35, 36). Tyler et al (22) demonstrated TGF-b1 mRNA and protein in periapical cysts and granulomas and assigns it a role in the repair process. Piattelli et al (20) observed a higher expression of TGF-b1 in odontogenic keratocysts than in RCs and DCs and associated this fact to the higher growth potential of keratocysts by intense cell proliferation, which would be regulated by TGF-b1. Gazivoda et al (27) added IL-10 and TGF-b to inflammatory periapical lesion cell cultures and evaluated the synthesis of proinflamma1296
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tory cytokines. They showed that both IL-10 and TGF-b were able to inhibit the production of IL-1, TNF-a, and IL-8 (bone resorption factors), with TGF-b showing the higher immunosuppressive activity. However, despite these conflicting findings, a high expression of TGF-b1 during healing of bone fractures has been observed, suggesting its involvement in the bone repair process (37–39). In a literature review, Janssens et al (38) analyzed 39 studies that investigated the effect of TGF-b1 on bone tissue, and they concluded that TGF-b1 promotes the healing of bone fractures and bone formation in vivo. They reported that TGF-b1 is a growth factor that can be considered for use as an osteogenic agent in bone fractures or as an adjunct in excessive bone resorption observed in osteoporosis. Furthermore, TGF-b1 regulates a great number of biological processes such as proliferation, survival, differentiation, recognition, apoptosis, cell migration, and production of extracellular matrix, thus having important functions in immune responses, angiogenesis, wound healing, and bone metabolism. The tendency toward a greater expression of TGFb in the fibrous capsule of DCs found in this study may be related to increased angiogenesis, which was already discussed in a previous study (24). Two studies using primary bone marrow cell cultures (14, 40) and another study using murine monocytes (41) showed the induction of osteoclastogenesis by TGF-b. In the last study, the authors observed high levels of RANKL, M-CSF, and TGF-b, which were associated with an increase of osteoclast development from preosteoclast cells/monocytes shown by the presence of tartrate-resistant acid phosphatase in multinucleated cells and the expression of other osteoclast-specific markers such as calcitonin receptor and vitronectin receptor. According to these authors, TGF-b acts on preosteoclasts, promoting the differentiation of these cells into osteoclasts. In the present study, no significant difference was observed in the immunoexpression of TGF-b between RCs and DCs in the epithelia or capsule. The presence of TGF-b1 in odontogenic cysts assumes a compensatory mechanism by which the inflammatory response acts in the repair and remodeling of bone-injured tissue (8, 22). We found no statistically significant difference of TGF-b and IFN-g in RCs and DCs and believe that the balance of these proteins observed in immunostaining may be favor an osteolytic activity as well as an osteogenic activity in the odontogenic cysts. These antagonistic actions may depend on the secretion of other proteins such as RANKL, VEGF, matrix metalloproteinases (MMPs), and proinflammatory cytokines such as IL-1 and TNF-a that should be analyzed together for a more effective JOE — Volume 40, Number 9, September 2014
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Acknowledgments The authors deny any conflicts of interest related to this study.
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