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Receptor activator of NF-κB ligand and osteoprotegerin expression in chronic apical periodontitis: possible association with inflammatory cells FAN Rong, SUN Bin, ZHANG Cheng-fei, LÜ Ya-lin, XUAN Wei, WANG Qian-qian and YIN Xing-zhe Department of Stomatology, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China (Fan R, Lü YL and Xuan W) Department of Special Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, China (Fan R, Zhang CF, Wang QQ and Yin XZ) Department of Oral and Maxillofacial Surgery, School of Stomatology, Capital Medical University, Beijing 100050, China (Sun B) Comprehensive Dental Care, Endodontics, Faculty of Dentistry, the University of Hong Kong, Hong Kong SAR, China (Zhang CF) Correspondence to: Dr. ZhANG Cheng-fei, Area of Endodontics, Comprehensive Dental Care, Faculty of Dentistry, the University of Hong Kong HKSAR, China (Email: [email protected]) Key Words: apical periodontitis; receptor activator of nuclear factor kappa B ligand; osteoprotegerin; inflammation; bone resorption; immunohistochemistry Background Receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators (RANKL and OPG) and inflammatory cell infiltration in chronic periapical periodontitis. Methods The samples of chronic periapical lesions (n = 40) and healthy periapical tissues (n = 10) were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analysed the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68. Results The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues (P<0.001). The number of RANKL-positive cells was higher in lesions with 1 severe inflammatory infiltration than in those with light inflammatory infiltration (P<0.05). Significantly increased RANKL expression was found with T lymphocytes (CD3+), macrophages (CD68+) and B lymphocytes (CD20+) infiltration (P<0.05). No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration. Conclusions RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption. Chronic apical periodontitis occurs as a result of local inflammatory response, which is mediated by infiltrated inflammatory cells and their products.1 The inflammatory process ultimately results in periapical bone resorption, which is the unique function of osteoclasts.2 Receptor activator of NF-κB ligand (RANKL), its receptor, RANK, and osteoprotegerin (OPG) play crucial roles in regulating the differentiation, activation and survival of osteoclasts in both health and disease.3,4 RANKL induces bone destruction, and its natural decoy receptor, OPG protects against bone destruction by preventing the binding of RANKL with its receptor RANK.5,6 During normal bone remodeling, osteoblasts are thought to provide RANKL that stimulates bone resorption. Abnormal activation of the immune system leads to bone destruction in diseases such as rheumatoid arthritis and periodontitis.3,4 Osteoimmunology is an emerging field of research dedicated to investigating the interactions between the immune and skeletal systems.7 Chronic periodontitis share some of the features of periapical periodontitis.1,8 It is evident that the major sources of RANKL mediating periodontal bone destruction in vivo are lymphocytes and macrophages. 4,9 RANKL and OPG have been recently shown to play critical roles in periradicular bone destruction.10,11 However, the precise mechanisms of periapical inflammatory bone resorption remains unclear. Periradicular infiltration is mainly characterized by 2 macrophages, and T and B lymphocytes.12 These cells may be important stimulators of osteroclastogenesis. It is generally accepted that T lymphocytes have multiple biological activities and have received considerable attention because of its central role in the concept of osteoimmunology.7,13,14 The role of macrophages in RANKL-induced bone resorption is controversial. High RANKL levels were related to the monocyte-macrophage activity in six periapical granulomas.15 Conversely, other reports revealed that activated macrophages do not express RANKL in vivo.16 Data are accumulating in support of B cells involvement in bone resorption.4,17 However, there are still few reports discussing the effect of B lymphocytes on periapical bone loss. It is general believed that the balance between RANKL and OPG is important for regulating osteoclastogenesis.5 The relative ratio of RANKL/OPG may also correlate with the clinical severity of periodontitis.18 To date, little information is available about whether the RANKL/OPG ratio is related to the severity of periapical inflammation. Accordingly, the present study aimed to investigate the expression of RANKL, OPG and T, B lymphocytes and macrophages immunohistochemically in periapical lesions, to evaluate whether the level of RANKL or RANKL/OPG ratio is related to inflammatory cells infiltration. MATERIAL AND METHODS Tissue Preparation This study obtained institutional review board approval by the Ethical Committee of Peking University Health Science Center. A total of 50 subjects were included in this study. Written and informed consents from all patients were obtained before sample collection. The diseased tissue samples comprised forty chronic periapical lesions. The samples were obtained from 40 patients, aged 16-60 (average 37) years, subjected to periapical surgery and curettage of the tissue as part of the clinical treatment. All patients had radiographic evidence of periapical lesions and disappearance of the periodontal 3 ligament space. The control group comprised ten samples of clinically healthy apical periodontal ligament obtained from permanent premolars extracted for orthodontic purposes from 10 subjects, (age, 18-23 years) presenting good oral health. All these premolars were with the completion of root development and without periapical pathosis. The periodontal ligament was scraped off the roots with a sterile blade. All patients were negative for systemic diseases and were not under antibiotics for the last 2 months. All samples were immediately fixed in 10% neutral buffered formalin overnight and embedded in paraffin. Immunohistochemistry EnVision 2-step immunoperoxidase method was applied to detect the expression of RANKL, OPG, CD3 (T lymphocyte marker), CD20 (B lymphocyte marker) and CD68 (macrophage marker). Consecutive sections were cut and stained using Envision peroxidase procedure (Dako, Glostrup, Denmark), according to the manufacturer's protocol. Briefly, 4-μm thick sections were deparaffinized and dehydrated followed by antigen retrieval, which was performed by autoclave in 10 mM citrate buffer (pH=6.0) for 10min (120℃, 2 atm). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min. After rinsing 3 times with PBS, sections were incubated for 30 min at room temperature (CD3, CD20 and CD68) or at 4°C overnight (RANKL and OPG) with the primary antibodies. Primary antibodies used in this study were as follows: anti-OPG antibodies (mAb8051; R&D Systems, Minneapolis, MN, USA; dilution 1:300), anti-RANKL antibodies (mAb626; R&D Systems; dilution 1:100), rabbit polyclonal anti-CD3 (Neomarkers, Fremont, CA; dilution 1:200) to detect T lymphocytes, rabbit polyclonal anti-CD20 (Neomarkers; dilution 1:400) to detect B lymphocytes, and mouse monoclonal anti-CD68 (Neomarkers; dilution 1:50) to detect macrophages. The sections were subsequently incubated with a polymer-peroxidase complex and 3,3′-diaminobenzidine (DAKO), and counterstained with haematoxylin. Negative controls with the omission of the primary antibody were performed for each case. 4 Cell Counting One section from each periapical lesion specimen was stained with haematoxylin and eosin for histologic analysis and grading of inflammation prior to immunohistochemistry. According to Tsai et al19, each specimen was graded at ×200 magnification as: grade - light, inflammatory cells less than 1/3 per field; grade moderate, inflammatory cells between 1/3 and 2/3 per field; and grade - severe, inflammatory cells more than 2/3 per field. The number of positively stained cells for RANKL or OPG was counted in 10 consecutive microscopic high-power fields (×400). According to Liapatas et al20, immunohistochemical reactivity for CD3, CD20 and CD68 was classified into five categories according to the percentage of positively staining cells as follows: 0= no staining; 1= light, less than 10%; 2= moderate, 10-25%; 3= intense, 25-50%; and 4= very intense, more than 50%. Statistical Analysis Data were expressed as mean ± standard deviation (SD). Differences among groups were assessed using Student’s t test or one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05. All data were analyzed by SPSS (Ver. 13.0, SPSS Inc., USA). RESULTS All periapical lesions showed positive immunoreactivity to RANKL and OPG (Figure 1A, C) and showed various degrees of inflammatory cell infiltration. RANKL protein was mainly in the nucleus of the lymphocytes, plasma cells, macrophage-like cells, epithelial cells and fibroblast-like cells. OPG protein was found mostly in the cytoplasm of the oral epithelium and inflammatory cells. RANKL and OPG were also expressed in the samples of normal controls (Figure 1B, D). The presence of CD3, CD20, CD68 antigens varied from light to intense in all periapical lesion specimens (Figure 2). The normal control subjects showed no or minimal inflammatory 5 infiltration. Negative controls were obtained by replacing the primary antibodies by PBS showing no stained cells. As shown in Table 1, Periapical lesion tissues had a significantly higher number of RANKL-positive cells (P < 0.001). There were no differences in OPG expression between periapical lesions and controls ( P > 0.05). Periapical lesion tissues showed a significantly higher ratio of RANKL/OPG ( P < 0.001). The number of RANKL-positive cells was significantly higher in periapical lesions with servere inflammation than those with light inflammation ( P < 0.05) (Table 2). There was a significant association between the number of RANKL-positive cells and the degree of CD3, CD20 and CD68 infiltration, respectively ( P < 0.05) (Table 3). Moreover, the ratio of RANKL/OPG did not show association with the degree of periapical inflammation, as well as CD3+, CD20+ and CD68+ infiltration ( P > 0.05) (Table 4, 5). DISCUSSION In the present study, the number of RANKL-positive cells, as well as the RANKL/OPG ratio in periapical lesions were significantly higher than those in healthy periapical tissues. The results are consistent with previous studies.11,14 RANKL expression was also detected in some healthy periapical tissues. Such findings, in accordance with Menezes et al11, may account for a regulatory role on osteoclasts to maintain a normal bone homeostasis. Furthermore, it could imply that only the existence and abundance of RANKL might not always lead to bone resorption. It is suggested whether bone resportion or bone formation occurs depends critically on the RANKL/OPG ratio, rather than a single value of RANKL or OPG.5 The number of RANKL-positive cells was found to increase with the grade of periapical inflammation, suggesting a clear positive relation between high RANKL expression and leukocytes activities during periapical bone loss. Furthermore, the number of RANKL-positive cells was found to be closely related to the degree of the main inflammatory cells, including T, B lymphocytes and macrophages. Lesions with intense infiltrated T cells showed a significantly higher RANKL expression than those 6 with light or moderate infiltrated T cells. T cells may play an important role not only as an initial trigger but also as a constant stimulator of bone resorption.21 In pathological conditions, the effects of T cells on osteoclastogenesis depend on a dynamic balance between positive and negative factors that they express.21 T help type 1 (Th1) immune response is involved in the progession of periapical lesion and bone destruction, whereas Th2 cytokines are more significant for the restriction of immune mechamisms.13,14 The balance between Th1 and Th2 cytokines may determine the overall biological events in the lesions.14 Previous studies in periodontitis have elucidated that activated CD4+ T cells express RANKL in vivo and activated CD8+ T cells suppress osteoclastogenesis in vitro.22,23 Further studies should be done to evaluate the roles of T-lymphocyte subsets in periapical bone loss, importantly, to identify the osteoclastogenic T-cell subset. In a recently work, most of RANKL+ cells were fibroblastic, but few of them were T cells in vitro.16 Other studies have also shown that gingival fibroblasts and periodontal ligament cells can be stimulated to produce RANKL via microbial challenge or contact with bacterial products.9 A significant association was found between the number of RANKL-positive cells and the degree of infiltrated macrophages. Our findings are in agreement with several studies.11,24,25 Macrophages may also participate in osteoclast formation, either by producing proinflammatory cytokines or by serving themselves, as osteoclast precursors.9,26 However, it is not sure wheather these cells were indeed the source of this RANKL or merely binding RANKL derived from other cells, because macrophages may also express RANK. Yoshinaga et al27 proposed no report has clearly shown that macrophages produce RANKL in vitro. Despite some controversies, the current consensus is that activated macrophages would participate in the formation and perpetuation of the periapical lesion and bone destruction. Further studies are needed to clarify this issue. Accumluating evidence indicate that B cells not only produce bacteria-reactive IgG, but also contribute to pathogenic bone resportion.9,28 In periodontal diseases, B and T cells are proved to be the mainly celluar source of RANKL in vivo.4 B cells may also 7 participate in osteoclast formation, either by expressing RANKL or by serving themselves, as osteoclast progenitor cells.29 Morever, osteoblasts support commitment and differentiation of all stages of B cells development.28 However, what is often overlooked is the close relationship between B cells and bone cells. Only one report was found discussing the potential role of B cells in RANKL-mediated periapical bone resorption, showing that lymphocytes were the predominant leukocyte cells, but monocytes and dendritic cells were mainly responsible for synthesizing RANKL in six periapical granulomas.15 Taken together, close relationships were found between the number of RANKL-positive cells and the degree of macrophages and T, B lymphocytes infiltration respectively, suggesting that these leukocytes may be the three most likely sources of RANKL in the periapical bone resorption. However, as a limitation of the immunohistochemistry technique, we were unable to definitively identify the cellular sources of RANKL. And not all inflammatory cells in periapical lesions are in a state of activation.26 Further studies should be carried out to precisely identify the distribution of RANKL-expressing cells. In the present study, the ratio of RANKL/OPG is not directly related to periapical inflammatory infiltration. The results are not unexpected, and similar observations were described by Geusens et al.30 RANKL-mediated osteoclastogenesis is not responsible for all pathological bone destruction. Other cytokines, such as interleukin-1(IL-1) and tumor necrosis factor α (TNF-α) can promote osteoclast formation by a fully or partilly independent-RANKL pathway.31 Our results can be further explained by a recent study that the RANKL/OPG ratio was associated with the progressive or stable nature of periapical granulomas.32 The progression and expansion of the disease and the occurance of bone destruction are initially trigged by the abnormal host immune response determined by inflammatory cells. Inflammatory infiltration in the periapex spaces seems to be a critical step in both immune responses and bone destruction. Although inflammation and osteoclastogenesis are different processes in the periapical region, proinflammatory cytokines act as major co-factors in osteoclast formation and activation. The outcomes of these pathological events are 8 determined by the balance between pro- and anti-inflammatory cytokines.8 Clinically, it is unlikely that periradicular bone destruction would occur in the absence of inflammation, but inflammation may occur in the absence of bone destruction. In conclusion, the expression of RANKL, but not the RANKL /OPG ratio, was significantlly associated with the grade of periapical inflammation. Morever, the expression of RANKL, but not the RANKL/OPG ratio, was also increased with the severity of macrophages or T-, B- lymphocytes. Our findings implied that RANKL appears to be closely related to periapical inflammatory infiltrates, and the relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption. References 1. Nair PN. Apical periodontitis: a dynamic encounter between root canal infection and host response. Periodontol 2000 1997;13:121-148. 2. Teitelbaum SL. Bone resorption by osteoclasts. Science 2000;289:1504-1508. 3. Haynes DR, Crotti TN, Loric M, Bain GI, Atkins GJ, Findlay DM. Osteoprotegerin and receptor activator of nuclear factor kappaB ligand (RANKL) regulate osteoclast formation by cells in the human rheumatoid arthritic joint. Rheumatology 2001;40:623-630. 4. Kawai T, Matsuyama T, Hosokawa Y, Makihira S, Seki M, Karimbux NY, et al. B and T lymphocytes are the primary sources of RANKL in the bone resorptive lesion of periodontal disease. Am J Pathol 2006;169:987-998. 5. Hofbauer LC, Heufelder AE. Role of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin in bone cell biology. J Mol Med 2001;79:243-253 6. GENG DC, XU YZ, YANG HL, ZHU GM, WANG XB, et al. Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis. Chin Med J 2011;124:586-590. 7. Arron JR, Choi Y. Bone versus immune system. Nature 2000;408:535-536. 8. Sila TA, Garlet GP, Fukada SY, Silva JS, Cunha FQ. Chemokines in oral inflammatory diseases: apical periodontitis and periodontal disease. J Dent Res 2007;86:306-319. 9. Liu YC, Lerner UH, Teng YT. Cytokine responses against periodontal infection: protective and 9 destructive roles. Periodontology 2000 2010;52:163-206. 10. Sabeti M, Simon V, Kermani Y, Valles Y, Rostein I. Detection of receptor activator of NF-κB ligand in apical periodontitis. J Endod 2005;31:17-18. 11. Menezes R, Bramante CM, da Silva Paiva KB, Letra A, Carneiro E, Fernando Zambuzzi W, et al. Receptor activator NFκB-ligand and osteoprotegerin protein expression in human periapical cysts and granulomas. Oral Surg Oral Med Oral pathol Oral Radiol Endod 2006;102:404-409. 12. Matsuo T, Ebisu S, Shimabukuro Y, Ohtake T, Okada H. Quantiative analysis of immunocompetent cells in human periapical lesions: correlations with clinical findings of the involved teeth. J Endod 1992;18:497-500. 13. Ihan Hren N, Ihan A. T lymphocyte activation and cytokine expression in periapical granulomas and radicular cysts. Arch Oral Boil 2009;54:156-161. 14. Fukada SY, Silva TA, Garlet GP, Rosa AL, da Silva JS, Cunha FQ. 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. 15. Vernal R, Dezerega A, Dutzan N, Chaparro A, León R, Chandía S, et al. RANKL in human periapical granuloma: possible involvement in periapical bone destruction. Oral Dis 2006;12:283-289. 16. Kawashima N, Suzuki N, Yang G, Ohi C, Okuhara S, Nakano-Kawanishi H, et al. Kinetics of RANKL, RANK and OPG expressions in experimentally induced rat periapical lesions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:707-711. 17. Hayer S, Polzer K, Brandl A, Zwerina J, Kireva T, Smolen JS, et al. B-cell infiltrates induce endosteal bone formation in inflammatory arthritis.J Bone Miner Res 2008;23:1650–1660. 18. Bostanci N, Ilgenli T, Emingil G, Afacan B, Han B, TÖz H, ea al. Gingival crevicular fluid levels of RANKL an OPG in periodontal diseases : implications of their relative ratio. J Clin periodontal 2007;34:370-376. 19. Tsai CH, Weng SF, Yang LC, et al. Immunohistochemical localization of tissue-type plasminogen activator and type I plasminogen activator inhibitor in radicular cysts. J Oral Pathol Med 2004;33:156-161. 20. Liapatas S, Nakou M, Rontogianni D. Inflammatory infiltrate of chronic periradicular lesions: an immunohistochemical study. Int Endod J 2003;36:464-471. 10 21.Takayanagi H. Inflammatory bone destruction and osteoimmunology. J Periodont Res 2005;40:287-293. 22. Choi Y, Woo KM, Ko SH, et al. Osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8+ T cells. Eur J Immunol. 2001;31:2179-2188. 23. Vernal R, Dutzan N, Hernández M, et al. High expression levels of receptor activator of nuclear factor-kappa B ligand associated with human chronic periodontitis are mainly secreted by CD4+ T lymphocytes. J Periodontol. 2006;77:1772-1780. 24. Crotti T, Smith MD, Hirsch R, Soukoulis S, Weedon H, Capone M, et al. Receptor activator NFκB ligand (RANKL) and osteoprotegerin (OPG) protein expression in periodontitis. J Periodontal Res 2003;38:380-387. 25. Crotti TN, Smith MD, Findlay DM, Zreiqat H, Ahern MJ, Weedon H, et al. Factors regulating osteoclast formation in human tissues adjacent to peri-implant bone loss: expression of receptor activator NFκB, RANK ligand and osteoprtogerin. Biomateials 2004;25:565-573. 26. Metzger Z. Macrophages in periapical lesions. Endod Dent Traumatol 2000;16:1-8. 27. Yoshinaga Y, Ukai T, Abe Y, Hara Y. Expression of receptor activator of nuclear factor kappa B ligand relates to inflammatory bone resorption, with or without occlusal truma, in rats. J Periodont Res 2007;42:402-409. 28. Horowitz MC, Fretz JA, Lorenzo JA. How B cells influence bone biology in health and disease. Bone 2010;47:472-479. 29. Manabe N, Kawaguchi H, Chikuda H, Miyaura C, Inada M, Nagai R, et al. Connection between B lymphocyte and osteoclast differentiation pathways. J Immunol 2001;167:2625-2631. 30. Geusens PP, Landewé RBM, Garnero P, Chen D, Dunstan CR, Lems WF, et al. The ratio of circulating osteoprotegerin to RANKL in early rheumatoid arthritis predicts later joint destruction. Arthritis Rheum 2006;54:1772-1777. 31. Jiang J, Li H, Fahid FS, Filbert E, Safavi KE, Spangberg LS, et al. Quantitative analysis of osteoclast-specific gene markers stimulated by lipopolysaccharide. J Endod 2006;32:742-746. 32. Menezes R, Garlet TP, Letra A, Bramante CM, Campanelli AP, Figueira Rde C, et al. Differential patterns of receptor activator of nuclear factor kappa B ligand/osteoprotegerin expression in human periapical granulomas: possible association with progressive or stable 11 nature of the lesions. J Endod 2008;34:932-938. Figure 1. Immunohistochemical analysis of RANKL and OPG expression in chronic periapical lesions and healthy periapical tissues (original magnification × 400). 1A: RANKL protein in periapical lesions. 1B: RANKL protein in healthy periapical tissues. 1C: OPG protein in periapical lesions. 1D: OPG protein in healthy periapcial tissues (arrows) Figure 2. Immunohistochemical staining for major inflammatory cells in chronic periapical lesions (original magnification × 400). 2A: CD3 marker for T lymphocytes. 2B: CD20 marker for B lymphocytes. 2C: CD68 marker for macrophages (arrows). Table 1. the semiquantitative analysis of lesion and control groups immunostained for RANKL and OPG Groups RANKL OPG Lesions ( n = 40 ) 42.03 ± 8.07 * 27.08 ± 4.56 1.57 ± 0.25* Controls ( n = 10 ) 13.61 ± 3.54 24.17± 4.58 0.48 ± 0.24 * P < 0.001, compared with the control group 12 RANKL/OPG ratio Table 2. The number of RANKL-positive cells and the ratio of RANKL/OPG according to the grade of inflammation The grade of The number of The ratio of inflammation RANKL-positive cells RANKL/OPG light (n=8) 35.74 ± 9.02 * 1.60 ± 0.26 moderate ( n = 17 ) 42.08 ± 8.07 1.52 ± 0.21 severe 45.34 ± 5.64 1.61 ± 0.15 ( n = 15 ) * P < 0.05 compared with severe inflammation. Table 3. The number of RANKL-positive cells and the ratio of RANKL/OPG according to the degree of inflammatory cells The degree of inflammatory cells T lymphocytes Light The number of RANKL-positive cells The ratio of RANKL/OPG (CD3) (n=9) 39.49 ± 8.84 * 1.56 ± 0.17 Moderate ( n = 20 ) 40.25 ± 7.82 † 1.53 ± 0.23 Intense 47.35 ± 5.62 1.64 ± 0.17 ( n = 11 ) B lymphocytes(CD20) Light (n=9) 36.16 ± 8.14 * 1.55 ± 0.27 Moderate ( n = 19 ) 41.96 ± 8.13 1.57± 0.21 Intense ( n = 12 ) 46.55 ±4.88 1.58 ± 0.14 Macrophages (CD68) 13 Light ( n = 12 ) 37.59 ± 9.33 * 1.55 ± 0.28 Moderate ( n = 18 ) 42.93 ± 7.05 1.53 ± 0.18 Intense ( n = 10 ) 45.74 ± 6.19 1.66 ± 0.10 * P < 0.05, significant difference between light and intense staining † P < 0.05, significant difference between moderate and intense staining