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Abstract
Background: Oral lichen planus is a T-cell-mediated chronic inflammatory disease affecting approximately 1% to 2% of the population, the etiology of which is currently unknown. The objectives of this study were to observe if senescence occurs in oral lichen planus, through the assessment of the immunohistochemical expression of a novel marker for senescence called Senescence marker protein-30 or regucalcin, and compare the expression to that in oral lichenoid reaction and non-specific inflammation. Subjects and Methods: The study material consisted of 30 cases of oral lichen planus, 15 cases of oral lichenoid reaction and 15 cases of non-specific inflammation. The number of positive cells in ten randomly selected high power fields were counted in the epithelium and the connective tissue separately and the mean was determined. Results: Mann–Whitney U test was used to statistically analyze if there was any significant difference in the expression of Senescence marker protein-30 between oral lichen planus, oral lichenoid reaction and non-specific inflammation. Even though a greater expression was seen in the oral lichen planus cases than oral lichenoid reaction, the difference in both the epithelium and connective tissue was not statistically significant. Conclusion: This study shows that in addition to the already known mechanisms like apoptosis and increased cell proliferation rates, the activated T-lymphocytes may also trigger a senescent change in the cells of oral lichen planus. As with the other mechanisms, this is also seen only in a small proportion of the cases.
Keywords: Cellular senescence, oral lichen planus, oral lichenoid reaction, regucalcin, senescence marker protein- 30
How to cite this article: Introduction 
A British physician, Erasmus Wilson is credited with the first description of the mucocutaneous lesion, lichen planus. Being somewhat common in occurrence, it plagues about 1%–2% of the general population. In 20%–30% of the cases, oral lesions may be exclusively evident, but in 50%–70% of the cases, it occurs in conjunction with dermatological manifestations.[1]
Though by far, etiology of oral lichen planus (OLP) remains unknown, both antigen-specific and nonspecific mechanisms are thought to play a role. Presently available data suggests that the etiology is an altered cell-mediated immunity with exogeneous and endogenous factors acting as a trigger, as a result of which the body's response to autoantigens is faulty, ultimately resulting in antigen-specific keratinocyte killing by cytotoxic T cells, most of which are CD8+.[2],[3],[4]
A series of complex molecular mechanisms are initiated which is focused on arresting the cell cycle for DNA repair, thus inducing apoptosis so that the cells with damaged DNA are eliminated. It has also been demonstrated that epithelial cells in OLP frequently respond with increased proliferation rates. Apoptosis in lichen planus has been studied previously.[5]
Recent evidence has shown that epithelial cells and inflammatory cells in OLP may respond to DNA damage by undergoing senescence, apart from apoptosis, whereby the cells remain viable but display characteristic changes in their morphology, physiology and gene expression, and are unable to divide. Thus, cellular senescence induces irreversible growth arrest. Adopting a senescent phenotype prevents a cell from undergoing apoptosis.[6],[7]
Unlike apoptosis, senescence in lichen planus has rarely been investigated. The studies which did investigate senescence was with p21, which is a cell cycle arrest marker rather than a direct marker of senescence.[5]
This study attempts to investigate senescence in oral lichen planus using Senescence marker protein-30 immunohistochemical marker. Senescence marker protein-30 (SMP-30) or regucalcin is a novel marker for senescence that has not been investigated so far in lichen planus. It was initially identified in the liver of aging rats. SMP-30 has also an anti-apoptotic action, as it prevents cell death caused by intracellular accumulation of calcium by enhancing plasma membrane calcium pumping activity.[8],[9]
The objectives of the study was to assess the expression of Senescence marker protein-30 in the epithelial and inflammatory component of oral lichen planus and to compare to that in oral lichenoid reaction.
Subjects and Methods Material for this study consisted of formalin-fixed, paraffin-embedded, specimens of diagnosed cases of 30 oral lichen planus, 15 oral lichenoid reaction and nonspecific inflammation. [Table 1] The diagnosis was further confirmed in H and E sections, according to the histopathological criteria given by WHO. The archival tissues were retrieved from the Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences, Virajpet and from The Oxford Dental College And Hospital, Bangalore. The method was immunohistochemistry.
Paraffin sections of 4 μm thickness were taken and mounted on 3-aminopropyl tri ethoxysilane (APES) coated slides (Biogenex Pvt. Ltd, Hyderabad, India) which were then incubated for one hour at 60°C. The sections were dewaxed in 2 changes of xylene each for 15 min and rehydrated through a series of graded alcohols and distilled water.
Heat-induced epitope retrieval method was followed for antigen retrieval. Blocking of endogenous peroxide was done by flooding the slides with 3% hydrogen peroxide for 5 min, followed by washing in 2 changes of tris buffered saline for 5 min each.
Secondary detection system used was the Novolink mini polymer kit (Leica biosystems Newcastle Ltd., UK) which consisted of the peroxide, protein and post-primary block, secondary antibody, DAB chromogen, DAB substrate buffer and Mayers haematoxylin. Protein block was used for 5 min to block non-specific reaction with other tissue antigens. The slides were then washed in 2 changes of tris buffered saline for 5 min each and the sections were then flooded with anti-RGN primary antibody (Sigma Aldrich, St. Louis, US) diluted with large volume UltraAb diluent (Thermoscientific Pvt Ltd., USA) in the ratio of 1:600 for 45 min. This was followed by two changes of tris buffered saline wash for 5 min each, to remove unbound antibodies.
Post-primary block was used for duration of 30 min to enhance the reaction between primary and secondary antibodies, and the slides then taken through 2 changes of tris-buffered saline.
The sections were then flooded with secondary antibody for 30 min which detects any tissue bound primary antibody. This was followed with a tris buffered saline wash.
DAB chromogen was diluted with DAB substrate buffer in the ratio 1:20 and the slides were flooded for 8 min and a visible brown precipitate developed at the antigen site. The slides were then washed in tris buffered saline and counterstained with Mayer's hematoxylin for 1 min and then coverslipped with DPX as mountant.
Sections of rat kidney were used as positive control [Figure 1] and sections which were taken through the immunohistochemistry procedure, but in which SMP-30 primary antibody was not added were used as negative control.
Figure 1: Photomicrograph showing expression of smp-30 in rat kidney (positive control)Cells were considered to be positive for the SMP-30 antibody if either nuclei or cytoplasm acquired a brown color. The number of positive cells in randomly selected 10 high power fields were counted separately for the epithelium and connective tissue and mean cell count was determined [Figure 2] and [Figure 3].[5]
Figure 2: Photomicrograph showing smp-30 expression in epithelium and connective tissue of oral lichen planus
Figure 3: Photomicrograph showing smp-30 expression in epithelium and connective tissue of olrThe data collected by the methods described above were subjected to statistical analysis using SPSS windows (version 17) software. Mann–Whitney U test was used to compare the means of the two independent populations as the number of scores obtained in the two groups were less than 20.
Results The results obtained are summarized in [Table 2], [Table 3], [Table 4], [Table 5], [Table 6].
Table 4: Mean cell expression of SMP-30 in epithelium and connective tissue of study groups
Table 5: Comparison between the mean expression of SMP-30 in the epithelium of oral lichen planus and oral lichenoid reaction using Mann-Whitney U test
Table 6: Comparison between the mean expression of SMP-30 in the connective tissue of oral lichen planus and oral lichenoid reaction using Mann -Whitney U test Discussion Cellular senescence, a process similar to apoptosis, induces irreversible growth arrest. Cells remain viable but display characteristic changes in their morphology, physiology and gene expression and are unable to divide. While senescence usually occurs after an extensive number of cell divisions, it also appears prematurely, independent of the number of cell divisions, in response to various physiological stresses such as DNA damage, oncogenic stress, and oxidative stress.[6],[10]
Studies have shown that interleukin-dependent inflammatory networks and in particular the pro-inflammatory cytokines Il-6 and IL-8 plays a central role in premature senescence induction. The induction of cellular senescence is further associated by a striking increase in the secretion of about 40-80 factors. This “senescence-associated secretory phenotype” (SASP) proteins include a wide range of growth factors, proteases, chemokines and cytokines including IL-6 and IL-8 which serves to reinforce the senescence growth arrest. Studies have also shown increased levels of NF-kappa B associated cytokines (IL-1α, IL-6, IL-8, TNF) in oral lichen planus patients.[11],[12],[13],[14] Thus lichen planus being a chronic inflammatory disease, there exists a possibility that other than proliferation or apoptosis, the cells in lichen planus may also show a senescent change.
Unlike apoptosis and cell proliferation, senescence in lichen planus has rarely been investigated. The studies which did investigate senescence was with p21, which is a cell cycle arrest marker rather than a direct marker of senescence.
Bascones et al.[5] found expression of p21, a protein that arrests cell cycle in G1 phase thus inducing differentiation and senescence in the basal layer of oral lichen planus by immunohistochemistry.
In this study, a novel marker for senescence called Senescence marker protein-30 (SMP-30) or regucalcin is used. It was initially identified in the liver of aging rats. The gene which encodes for SMP-30 is highly conserved among the different animal species and is shown to be down-regulated in an androgen-independent manner in senescent stages.[8],[15]
It has been widely accepted that intracellular Ca2+ is a key player in regulation of various cellular functions. The role of Ca2+ as a cell death trigger was first suggested by A. Fleckenstein et al.,[16] and has been subsequently emphasized by various studies. Recent research work has shown that cells deficient in type I Ins (1, 4, 5) P3 receptors, which would be resistant to apoptosis, can be made to enter the apoptotic pathway through increase of cytoplasmic Ca2+. Ca2+ mediated apoptosis can act through a myriad of potential targets such as calcineurin, nitric oxide synthase, endonucleases etc.[17]
SMP-30 has been found to suppress extracellular ATP-induced Ca2+ elevation by enhancing Ca2+ efflux across the plasma membrane, suggesting the importance of SMP30 for preventing cell death caused by apoptosis and resulting in the senescent phenotype.[9]
In this study, the immunohistochemical expression of Senescence marker protein-30 was assessed in 30 cases of archival material where the diagnosis of oral lichen planus was given. The diagnosis was verified according to the histopathological criteria set by WHO in 1978 from routine hematoxylin and eosin sections. 15 cases of oral lichenoid reaction and 15 cases of non-specific inflammation were also assessed for SMP-30 expression. The expression in epithelial and inflammatory component was assessed separately and the mean cell expression determined.
It has been proven that there is an increase in senescence occurring in T lymphocytes seen in peripheral blood smear of patients exposed to HCV infection and since a known association exists between HCV infection and lichen planus; the expression of SMP-30 in lymphocytes was separately assessed.[18],[19]
We found positive expression in 26.67% of the cases of oral lichen planus, which is in accordance with the findings of the study done by Bascones et al.[5] where they found positive expression of p21 marker in 32.1% of the cases.
Greater expression was seen in the basal and suprabasal layers of the epithelium, as was found by Bascones et al.[5] and in accordance with the known fact that basal cells are the target of attack in lichen planus.
As the number of overall values obtained were less than 20, non-parametric Mann–Whitney U test was used to statistically analyze if there was any significant difference in the expression of SMP-30 between oral lichen planus and oral lichenoid reaction. A value P < 0.05 was considered statistically significant.
None of the nonspecific inflammation cases showed a positive expression and was by default not included in the statistical analysis. Approximately 26.67% of the oral lichen planus cases showed a positive expression in the epithelium as compared to 6.66% of the lichenoid reaction. Even though a greater expression was seen in the oral lichen planus cases, the difference was not statistically significant with P = 0.118.
13.33% of the oral lichen planus cases showed positive expression in the connective tissue as compared to 6.66% of the lichenoid reaction cases and the difference was not statistically significant (P = 0.544). The cases which have shown expression in the connective tissue have also shown epithelial expression.
The fact that SMP-30 was seen only in cases of oral lichen planus and was not seen in cases of non-specific inflammation and only one case of lichenoid reaction emphasizes the fact that senescence is one of the mechanisms adopted by the keratinocytes against the inflammatory challenge posed by the activated T-lymphocytes. The non-specific inflammation and the inflammatory infiltrate in lichenoid reactions do not have an anti-keratinocytic action and hence senescence is not seen in these conditions.[20]
The adaptation of a senescent phenotype by the basal cells in oral lichen planus might prove to be beneficial, in that these cells are able to resist the lymphocytic attack and thereby maintain a pool of cells with replicative capabilities which would be necessary to aid in the possible regeneration of the epithelium, once the inflammatory challenge has subsided.[5]
The senescent phenotype may also help the basal keratinocytes which have undergone DNA damage as a result of inflammatory cytokines by giving an opportunity to undergo repair and maintain at least transiently the structural integrity of the epithelium.[5]
Considering that the senescence mechanism is an example of antagonistic pleiotropism, the scenario that the senescent cells with DNA damage may also escape the normal immune surveillance and at a later stage transform into a pre-malignant or malignant phenotype is also a possibility. Hence, to comment whether senescence in oral lichen planus is a beneficial or adversarial would be premature.[10]
An interesting finding of our study was that of SMP-30 positive senescent lymphocytes in the connective tissue of four cases of OLP. Scientific evidence of a possible link between HCV and OLP has been increasing in the past few years. Recent meta-analyses have shown HCV and oral lichen planus to be significantly associated. Studies have also proven that in patients with HCV induced hepatitis, lymphocytes in peripheral blood and in tissues have shown senescence.[18],[19] Hence, it would be reasonable to hypothesize that the proportion of oral lichen planus cases showing senescent lymphocytes could have been HCV infected, however being a retrospective study, we were not able to investigate this further.
It has also been proven that in many chronic inflammatory diseases such as rheumatoid arthritis, juvenile idiopathic arthritis, Wegener's granulomatosis, atherosclerotic coronary artery disease, and inflammatory bowel disease, persistent immune activation accelerates the replicative senescence of T- and B-lymphocytes.[21] Since lichen planus is also a chronic inflammatory disease, though not in the classical sense, this could also be another explanation for the lymphocytic senescence observed in the oral lichen planus cases.
The findings of our study should be taken with the caveat of the small sample size and the fact that only 26.67% of the cases of oral lichen planus showed senescence.
Studies on the other cellular mechanisms in oral lichen planus, such as apoptosis and cell proliferation, have also yielded equivocal results and each mechanism has been seen in around 30%–40% of all cases studied, possibly pointing to a cyclical or transient manifestation of these cellular mechanisms. It is quite possible that a coordinated concert of apoptosis to senescence and proliferation are at play in oral lichen planus.
The exact nature of the interactions between these cellular mechanisms may be further unraveled by properly designed sequential cell culture studies rather than by single-point observations as in our study.
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Conflicts of interest
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References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_864_21
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