|Year : 2016 | Volume
| Issue : 2 | Page : 140-145
Immunoexpression of interleukin-6 in drug-induced gingival overgrowth patients
Department of Periodontics and Implantology, Government Dental College, Chennai, Tamil Nadu, India
|Date of Web Publication||27-May-2016|
P R Ganesh
F-157, F-Block, Anna Nagar East, Chennai - 600 102, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: To analyze the role of proinflammatory cytokines in drug-induced gingival enlargement in Indian population. Aim: To evaluate for the presence of interleukin-6 (IL-6) in drug-induced gingival enlargement and to compare it with healthy control in the absence of enlargement. Materials and Methods: Thirty-five patients selected for the study and divided into control group (10) and study group (25) consisting of phenytoin (10); cyclosporin (10) and nifedipine (5) induced gingival enlargement. Gingival overgrowth index of Seymour was used to assess overgrowth and allot groups. Under LA, incisional biopsy done, tissue sample fixed in 10% formalin and immunohistochemically evaluated for the presence of IL-6 using LAB-SA method, Labeled- Streptavidin-Biotin Method (LAB-SA kit from Zymed- 2nd generation LAB-SA detection system, Zymed Laboratories, CA). The results of immunohistochemistry were statistically analyzed using Kruskaal–Wallis and Mann–Whitney test. Results: The data obtained from immunohistochemistry assessment shows that drug-induced gingival overgrowth (DIGO) samples express more IL-6 than control group and cyclosporin expresses more IL-6 followed by phenytoin and nifedipine. Conclusion: Increased IL-6 expression was noticed in all three DIGO groups in comparison with control group. Among the study group, cyclosporin expressed maximum IL-6 expression followed by phenytoin and nifedipine.
Keywords: Cytokines, drug-induced gingival overgrowth, fibrosis, immunohistochemistry, interleukin-6, tissue inhibitor of metalloproteinase
|How to cite this article:|
Ganesh P R. Immunoexpression of interleukin-6 in drug-induced gingival overgrowth patients. Contemp Clin Dent 2016;7:140-5
| Introduction|| |
Many systemic diseases require long-term medical management with drugs regardless of any adverse effects and manifestations. Gingival overgrowth is one of the known side effects of a few drugs administered over a prolonged period. The most common drugs causing this condition are immunosuppressants like cyclosporin, anticonvulsants like phenytoin and calcium channel blockers like nifedipine. Although the pharmacologic effects of these drugs differ widely, they all seem to affect the gingiva similarly, causing gingival overgrowth.
Several studies have reported that local, genetic, and systemic factors may contribute to the development of drug-induced gingival overgrowth (DIGO)., Studies regarding the pathogenesis of DIGO have revealed considerable information on connective tissue metabolism involved in DIGO., Many such studies have shown that elevated levels of various cytokines like interleukin-6 (IL-6), IL-1β, transforming growth factor-β (TGF-β); fibroblast growth factor and platelet-derived growth factor (PDGF) are present in DIGO and that these cytokines play a significant role in the pathogenesis of drug-induced gingival overgrowth.,,
IL-6 is a pleiotropic cytokine produced by a wide range of cell types including lymphocytes, monocytes, fibroblasts, and endothelial cells. The gingival fibroblast, the predominant cell type in the gingiva, is responsible for the synthesis and turnover of collagen and glycosaminoglycans in the extracellular matrix of the gingival tissues., IL-6 was found to stimulate gingival fibroblasts and play a major role in extracellular matrix synthesis., Elevated levels of IL-6 were also found in pulmonary and renal fibrosis and to be associated with scleroderma.,,
Several studies by various methodologies were conducted about pathogenesis of DIGO with the motive to control and prevent it with the aim of improving esthetics and restoring function., However, the complexities of the events that contribute to drug-induced gingival overgrowth are yet to be elucidated clearly. As just a handful of studies have investigated the role of IL-6 in DIGO and considering the paucity of studies in the Indian population on DIGO the present comparative drug effect study was undertaken.,, Thus, the objective of this study was to evaluate the involvement of IL-6 in DIGO by highly specific LAB-SA immunohistochemistry so as to assess the role of IL-6 in the pro-inflammatory processes involved in DIGO.
| Materials and Methods|| |
After obtaining the Ethical Clearance from the Institutional Ethical Review Board, patients attending the departments of neurology, cardiology, and transplant medicine and who were undergoing treatment with phenytoin; cyclosporin and nifedipine were screened for gingival overgrowth. From them, thirty-five patients were selected for the study and divided into two groups, the control and study groups. The control group of ten patients was from systemically healthy patients reporting for esthetic gingivoplasty procedures and crown lengthening procedures prior to restorations. The control patients had not been prescribed any drugs known to cause gingival overgrowth while for the study group apart from taking phenytoin, cyclosporin or nifedipine, these patients had not been prescribed any other drugs known to cause gingival overgrowth.
The study group consisted of groups A, B, and C.
- Group A: Phenytoin-induced gingival overgrowth (ten patients)
- Group B: Cyclosporine-induced gingival overgrowth (ten patients)
- Group C: Nifedipine-induced gingival overgrowth (five patients)
The study procedure was explained to all patients, and informed consent was obtained prior to the study to satisfy institutional ethical requirements. Thorough history taking and clinical examination assessing gingival overgrowth by using gingival overgrowth index (GOI) of Seymour was done in all patients. GOI scores 2 or 3 only were taken for study groups. All patients underwent routine hematological and biochemical investigations. All patients underwent scaling and obtained fitness from their regular physician prior to biopsy.
Gingival overgrowth index by Ellis and Seymour (2004)
Criteria for assessing gingival overgrowth
- No encroachment of interdental papilla onto tooth surface
- Mild encroachment of interdental papilla
- Moderate encroachment involving lateral spread of papilla across buccal tooth surface of less than one-quarter of tooth width
- Marked encroachment of papilla, i.e., more than one-fourth width. Loss of normal papilla form
The GOI of Seymour  is the index most commonly used for clinically  quantifying the overgrowth seen in DIGO. Marked on a scale of 0–3 it provides an easy reference to quantify the severity of overgrowth and also to plan the surgical intervention necessary for management of the overgrowth.
After anesthetizing the sample site using 2% lignocaine, gingival biopsy samples [Figure 1] from interdental papilla, gingival margin, and attached gingiva were obtained using a Bard-Paker blade no 15 [Figure 1]. The tissue samples were labeled, fixed in 10% neutral buffered formalin and embedded in paraffin.
Using a microtome, tissue sections of 4 µ were sectioned from the paraffin embedded, formalin fixed, and tissue blocks. Two slides were made from each tissue specimen, one slide for hematoxylin and eosin (H and E) staining and one slide for immunohistochemistry analysis for both control and study groups. All slides were given code numbers to mask the control and study groups so as to prevent bias.
The H and E staining was done to examine for gingival hyperplasia and fibrosis. Moreover, finally using the primary antibody IL-6 (IL-6 antibody from GeneTex, USA - rabbit polyclonal to human IL-6, dilution–1: 400) and the secondary antibody histostain plus kits for LAB-SA Detection system - From Zymed laboratories) immunohistochemical staining and evaluation with control slides stained with positive tissue control (esophageal carcinoma specimen containing maximum IL-6) and negative tissue control (specimen treated with a nonimmune serum instead of same concentration of primary antibody) were done. To prevent any examiner bias the results of immunohistochemistry were analyzed and graded for standardization by two independent oral pathologists for expression of IL-6 in the study and control samples based on severity of staining as per the IHC gradings for tissue samples.
| Results|| |
The statistical package SPSS PC+ (Statistical Package for Social Sciences, Ver. 14.01 IBM industries, Armonk, New York) was used for statistical analysis. The mean values and test of significance were obtained using the Kruskaal–Wallis (H test) and Mann–Whitney test.
The fibrosis and IL-6 positive staining was more in the study group when compared to control group. The cyclosporin group (Group B) (ten patients) were more positive for IL-6 in comparison to nifedipine group (Group C) (five patients) and Phenytoin group (ten patients) (Group A).
[Table 1] shows the mean values in control and study groups using Kruskaal–Wallis test and the P values showed statistical significance in the study group when compared to control group. [Figure 2] shows the graphical representation of data, wherein the study group (Groups A, B, C) (phenytoin, cyclosporin, nifedipine,) expresses more IL-6 expression in comparison to control group [Figure 3], [Figure 4], [Figure 5].
|Table 1: The mean value in control and study groups and their test of significance using Kruskall–Wallis test|
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|Figure 3: Photomicrograph (×10/×40) showing study Group A (phenytoin) taking up positive IHC staining for interleukin-6|
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|Figure 4: Photomicrograph (×10/×40) showing study Group B (cyclosporin) taking positive IHC staining for interleukin-6|
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|Figure 5: Photomicrograph (×10/×40) showing study Group C (nifedipine) taking positive IHC staining for interleukin-6|
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[Table 2] [Figure 6] shows the result of Mann–Whitney test to compare the control and study Group B (cyclosporin) and the test of significance (P< 0.05) showing that there is statistical significance which is very high in fibroblasts followed by blood vessels.
|Table 2: The mean and P value of Mann–Whitney U-test of control and study Group B (cyclosporin)|
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[Table 3] and [Table 4] [Figure 7] and [Figure 8] show the results of Mann–Whitney test to compare control with study Group A (phenytoin) and study Group C (nifedipine) respectfully. On comparison, the P values show that there is maximum significance of IL-6 staining in cyclosporin group followed by the phenytoin and nifedipine groups.
|Table 3: The mean and P value of Mann–Whitney U-test of control and study Group A (phenytoin)|
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|Table 4: The mean and P value of Mann–Whitney U-test of control and study Group C (nifedipine)|
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| Discussion|| |
Drug-induced gingival overgrowth remains the most widespread side effect of systemic medications on the periodontal tissues. Three major drug categories – the anti-convulsants, the calcium channel blocking agents and the immunosuppressants have been associated with gingival overgrowth with a reported prevalence rate of 90%. Incidence and prevalence studies have also reported on a wide variability in gingival response to these medications and other confounding variables and risk factors such as – age, gender, dose and duration of drug use, concomitant use of other medications, ability to maintain oral hygiene, role of local factors, and the genetically determined capacity of the host to deal metabolically with chronically administered drugs and the responsiveness of gingival fibroblasts to these drugs.,
The etiopathogenesis of drug-induced gingival overgrowth has been studied by various authors who have enlightened on the complex pathogenesis of DIGO and the various roles played by a range of fibrogenic cytokines like IL-6, and growth factors like PDGF, TGF-β and connective tissue growth factor (CTGF) in the connective tissue metabolism in gingival overgrowth.,,
Several studies ,, have reported that a key histopathological feature of drug-induced gingival overgrowth is a dramatic elevation in the expression of specific cytokines, especially IL-6 by cells within the gingival connective tissue.,, These reports are consistent with the findings of increased levels of IL-6 in other fibrotic diseases such as renal and pulmonary fibroses.,
Rakhi Sinha Morton and Anna Dongari Bagtzoglou reported  an enhanced IL-6 secretion in DIGO by Gingival Fibroblasts, thus highlighting the role of pro-fibrotic and pro-inflammatory cytokines in the pathogenesis of drug-induced gingival overgrowth.
Since very few studies have been done to assess the role of IL-6 in drug-induced gingival overgrowth in the Indian population, the present study was undertaken.,,
In this study, the IL-6 staining using IL-6 Polyclonal antibody is more in the study group than in the control group. There was statistical significance (P< 0.05) in the study group in concurrence with previous studies., The IL-6 staining was maximum in the cyclosporine group followed by the phenytoin and nifedipine groups similar to the study by Rakhi Sinha Morton and Anna Dongari Bagtzoglou. In their study, they state that the gingival fibroblasts respond to these drugs with an increased expression of IL-6. Williamson et al. reported that in DIGO there is an alteration in the cytokoine profile causing a dysregulation in the connective tissue turnover with a resultant accumulation of matrix components and fibroplasia.
Of all the cytokines playing a regulatory role in the gingival and periodontal connective tissues, IL-6 appears to play an autoregulatory role for both pro-fibrotic and pro-inflammatory cytokines., Furthermore, IL-6 targets the connective tissue cells like fibroblasts , and enhances connective tissue accumulation through increased production of tissue inhibitor of metalloproteinase , (TIMP) by fibroblasts without any effect on matrix metalloproteinase secretion ,, thus ultimately resulting in fibroplasia.
Since the pathogenesis of DIGO is attributed to alterations in the levels of both pro-inflammatory and pro-fibrotic cytokines like IL-6 and IL-1 in addition to growth factors like TGF-β and CTGF, further multifactorial studies with larger sample sizes are required to form a definitive conclusion on the role played by cytokines and growth factors in drug-induced gingival overgrowth.
| Conclusion|| |
This study was done to evaluate the role of inflammatory cytokines in the pathogenesis of DIGO as a way to prevent or control the overgrowth. The results of this study show clearly that the pro-inflammatory cytokine IL-6 expression was more in the DIGO groups than in the control group.
The extent of IL-6 expression was maximum in cyclosporin takers followed by those taking phenytoin and nifedipine. The gingival fibroblasts in all the study groups showed the most statistical significance along with nonspecific staining of blood vessels and epithelium which are of no statistical significance. Future studies may focus on increasing sample size, alternative drug therapy, combination drug therapy, GCF concentration, and also the quantum of drug necessary for induction of gingival overgrowth.
Even though much depends on genetic variability, fibroblast heterogeneity, and individual host response the fact that cytokine expression is elevated in the pathogenesis of DIGO can lead to development of anti-inflammatory modalities as a necessary treatment option in the prevention and management of gingival overgrowth. This study underscores the fact that prevention of cytokine expression by early periodontal therapeutic management and oral hygiene practices may lead to resolution of inflammation and subsequent prevention of DIGO. Hence, the role of a periodontist in early screening and co-management of systemic drug takers cannot be emphasized enough.
I wish to acknowledge with thanks the timely help given by Dr. T. S. S. Kumar, Dr. P. K. Saraswati and Dr. K. H. P. Shankar during my study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Rees TD, Levine RA. Systemic drugs as a risk factor for periodontal disease initiation and progression. Compend Contin Educ Dent 1995;16:20-42.
Thomason JM, Kelly PJ, Seymour RA. The distribution of gingival overgrowth in organ transplant patients. J Clin Periodontol 1996;23:367-71.
Tindall OP, Friskopp J. Drug induced gingival overgrowth – Review. Br Dent J 1987;184:305-8.
Callard R, George AJ, Stark J. Cytokines, chaos, and complexity. Immunity 1999;11:507-13.
Chae HJ, Ha MS, Yun DH, Pae HO, Chung HT, Chae SW, et al.
Mechanism of cyclosporine-induced overgrowth in gingiva. J Dent Res 2006;85:515-9.
Attila G, Kutuckler N. Crevicular fluid interleukin-1β, TNF-α, and Il-6 levels in patients with cyclosporin induced gingival overgrowth. J Periodontol 1998;69:784-90.
Dongari-Bagtzoglou AI, Ebersole JL. Increased presence of interleukin-6 (IL-6) and IL-8 secreting fibroblast subpopulations in adult periodontitis. J Periodontol 1998;69:899-910.
Hong HH, Trackman PC. Cytokine regulation of gingival fibroblast lysyl oxidase, collagen, and elastin. J Periodontol 2002;73:145-52.
Kishimoto T. The biology of interleukin-6. Blood 1989;74:1-10.
Narayanan AS, Meyers DF, Page RC. Regulation of collagen production in fibroblasts cultured from normal and phenytoin-induced hyperplastic human gingiva. J Periodontal Res 1988;23:118-21.
Trackman PC, Kantarci A. Connective tissue metabolism and gingival overgrowth. Crit Rev Oral Biol Med 2004;15:165-75.
Bartold PM, Haynes DR. Interleukin-6 production by human gingival fibroblasts. J Periodontal Res 1991;26:339-45.
Silver RM. Interstitial lung disease of systemic sclerosis. Int Rev Immunol 1995;12:281-91.
Lonnemann G, Engler-Blum G, Müller GA, Koch KM, Dinarello CA. Cytokines in human renal interstitial fibrosis. II. Intrinsic interleukin (IL)-1 synthesis and IL-1-dependent production of IL-6 and IL-8 by cultured kidney fibroblasts. Kidney Int 1995;47:845-54.
Feghali CA, Bost KL, Boulware DW, Levy LS. Mechanisms of pathogenesis in scleroderma. I. Overproduction of interleukin 6 by fibroblasts cultured from affected skin sites of patients with scleroderma. J Rheumatol 1992;19:1207-11.
Iacopino AM, Doxey D, Cutler CW, Nares S, Stoever K, Fojt J, et al.
Phenytoin and cyclosporine A specifically regulate macrophage phenotype and expression of platelet-derived growth factor and interleukin-1in vitro
and in vivo
: Possible molecular mechanism of drug-induced gingival hyperplasia. J Periodontol 1997;68:73-83.
James JA, Linden GJ. Drug-induced gingival overgrowth and class II major histo compatibility antigens. Transplantation 1998;57:1811-3.
Dongari-Bagtzoglou A, Lang N, McDoneel H T. Drug induced gingival overgrowth. Oral Surg Oral Med Oral Pathol 1993;76:543-8.
Morton RS, Dongari-Bagtzoglou AI. Regulation of gingival fibroblast interleukin-6 secretion by cyclosporine A. J Periodontol 1999;70:1464-71.
Ellis JS, Seymour RA, Taylor JJ, Thomason JM. Prevalence of gingival overgrowth in transplant patients immunosuppressed with tacrolimus. J Clin Periodontol 2004;31(2):126-31.
Thomason JM, Seymour RA, Rice N. The prevalence and severity of cyclosporin and nifedipine-induced gingival overgrowth. J Clin Periodontol 1993;20:37-40.
Bartold PM. Cyclosporine and gingival overgrowth. J Oral Pathol 1987;16:463-8.
Hassell TM, Hefti AF. Drug-induced gingival overgrowth: Old problem, new problem. Crit Rev Oral Biol Med 1991;2:103-37.
Myrillas TT, Linden GJ, Marley JJ, Irwin CR. Cyclosporin A regulates interleukin-1beta and interleukin-6 expression in gingiva: Implications for gingival overgrowth. J Periodontol 1999;70:294-300.
Williamson MS, Miller EK, Plemons J, Rees T, Iacopino AM. Cyclosporine A upregulates interleukin-6 gene expression in human gingiva: Possible mechanism for gingival overgrowth. J Periodontol 1994;65:895-903.
Hirano T. Interleukin 6 and its receptor: Ten years later. Int Rev Immunol 1998;16:249-84.
Kishimoto T, Taga T, Akira S. Cytokine signal transduction. Cell 1994;76:253-62.
Kent LW, Rahemtulla F, Hockett RD Jr., Gilleland RC, Michalek SM. Effect of lipopolysaccharide and inflammatory cytokines on interleukin-6 production by healthy human gingival fibroblasts. Infect Immun 1998;66:608-14.
Tüter G, Serdar MA, Yalim M, Gürhan IS, Balos K. Evaluation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 levels in gingival fibroblasts of cyclosporin A-treated patients. J Periodontol 2002;73:1273-8.
Yamada H, Nishimura F, Naruishi K, Chou HH, Takashiba S, Albright GM, et al.
Phenytoin and cyclosporin A suppress the expression of MMP-1, TIMP-1, and cathepsin L, but not cathepsin B in cultured gingival fibroblasts. J Periodontol 2000;71:955-60.
Modéer T, Domeij H, Andurén I, Mustafa M, Brunius G. Effect of phenytoin on the production of interleukin-6 and interleukin-8 in human gingival fibroblasts. J Oral Pathol Med 2000;29:491-9.
Romanos GE, Schröter-Kermani C, Hinz N, Herrmann D, Strub JR, Bernimoulin JP. Extracellular matrix analysis of nifedipine-induced gingival overgrowth: Immunohistochemical distribution of different collagen types as well as the glycoprotein fibronectin. J Periodontal Res 1993;28:10-6.
Sato T, Ito A, Mori Y. Interleukin 6 enhances the production of tissue inhibitor of metalloproteinases (TIMP) but not that of matrix metalloproteinases by human fibroblasts. Biochem Biophys Res Commun 1990;170:824-9.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4]