ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10015-2262 |
Role of Apoptosis in Inflammatory Gingival Lesions: A Histochemical Study
1-3Department of Periodontology, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Bengaluru, Karnataka, India
4-7Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Bengaluru, Karnataka, India
Corresponding Author: Kranti K Reddy, Department of Periodontology, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Bengaluru, Karnataka, India, Phone: +91 9880382393, e-mail: drkrantireddy@gmail.com
Received on: 03 May 2023; Accepted on: 04 June 2023; Published on: 22 August 2023
ABSTRACT
Aim: The regulation of inflammation and the host immune response relies significantly on apoptosis. It aids in tissue homeostasis, and a disruption of this is frequently linked to disease. The use of histochemical stains like hematoxylin and eosin (H&E) and Feulgen reaction for DNA can provide a simple and cost-effective method for the detection of apoptotic cells. The aim of this study was to analyze the expression of apoptosis in the gingival epithelium of gingivitis subjects and in patients with chronic periodontitis, using H&E and Feulgen reaction for DNA, aided by immunohistochemistry (IHC).
Materials and methods: A total of 20 gingival biopsies were harvested from gingivitis subjects (n = 10) and 10 subjects who suffered from chronic periodontitis (n = 10). On the day the samples were collected, a University of North Carolina (UNC) 15 periodontal probe was used to record the periodontal data which includes the bleeding index, plaque index, gingival index, probing depth, and attachment loss. Apoptotic cells were analyzed using Feulgen reaction for DNA and H&E under light microscopy. This was followed by immunohistochemical analysis of p53 and Bcl-2 biomarkers.
Results: Apoptotic cell count was higher in the chronic periodontitis group with a mean apoptotic index (AI) of 15.00 compared to gingivitis the group where the mean AI was 7.48. The mean difference was found to be −7.52, however the difference was not statistically significant (p = 0.18).
Conclusion: p53 plays a pivotal role in periodontal ligament cell homeostasis and seems to be upregulated in oral inflammatory diseases. Bcl-2 being an antiapoptotic marker was associated more with gingivitis as compared to chronic periodontitis.
How to cite this article: R DB, N SJ, Reddy KK, et al. Role of Apoptosis in Inflammatory Gingival Lesions: A Histochemical Study. World J Dent 2023;14(6):530-534.
Source of support: Nil
Conflict of interest: None
Keywords: Apoptosis, Chronic periodontitis, Gingivitis.
INTRODUCTION
Multiple species of gram-negative organisms are involved in the local and peripheral infection that implies inflammatory periodontal disorders.1 Neutrophil migration and consequent release of inflammatory mediators and cytokines constitute a part of the local host response to microorganisms, and these events appear to be crucial in the etiopathogenesis of periodontal disease.2 Both immune-mediated responses and the direct cytopathic effects of pathogens may be entailed in the mechanisms causing gingival tissue destruction, which are not well-understood. It has been speculated that apoptosis may be crucial in the development of periodontitis based on the immediate influence of bacteria in cell cultures. Cell death is an integral part of the tissue dynamics which are mediated through two processes; apoptosis and necrosis.3,4
Apoptosis is an important mechanism that is involved in maintaining normal homeostasis not only in the evolution of life forms but also in development. Apoptosis plays an important role in the pathogenesis of the periodontal disease.5 This form of cell death is important in both the inflammatory cells as well as cells of the periodontium. Programmed cell death or apoptosis is a normal physiologic process that contributes to maintaining tissue homeostasis. The process of apoptosis can be modulated by various stimuli, including hormones, cytokines, growth factors, bacterial or viral infections, and immune responses.6
Among other factors, the products of two genes that encode proteins p53 and Bcl-2 have been shown to play a fundamental regulatory role in apoptosis. Bcl-2 is a protein that belongs to the antiapoptotic family that can stop or lower cell death brought about by a range of factors.7 Antiapoptotic Bcl-2 proteins restrict the release of cytochrome c from the mitochondria, which starts the intrinsic death pathway. On the contrary, p53, a tumor-suppressor gene’s protein product, may initiate apoptosis when expressed. This protein is primarily believed to cause apoptosis in terminally differentiated cells, including inflammatory cells, and additionally plays a role in the regulation of tissue dynamics.8
There have been reports that a number of periodontal pathogens, including Porphyromonas gingivalis (P. gingivalis), Aggregatibacter actinomycetemcomitans, and Eikenella corrodens, cause cytotoxicity in different cellular elements of the periodontium.9 It has been proposed that apoptosis may be critical in periodontitis based on the direct cytopathic action of bacteria in cell cultures.
Moreover, a toxin from microbes triggers apoptosis in B lymphocytes found in periodontal tissue, while lipopolysaccharides (LPS), a prevalent component of gram-negative bacterial cell walls, enhance butyric acid-induced apoptosis in human peripheral blood mononuclear cells.10 Periodontal disorders have a pathogenesis that involves the induction of apoptosis in the host’s cells as a result of specific pathogens or their products. The apoptosis of the polymorphonuclear neutrophils (PMNs) may be delayed by bacterial phagocytosis or exposure to other bacterial components, such as LPS.11 In response to LPS, it showed that neutrophil apoptosis provided a signal to monocytes, altering their phenotype and causing the production of antiinflammatory cytokines whilst suppressing proinflammatory cytokines. Apoptosis mechanisms should play an important role in the elimination and renewal of periodontal cells.12 Despite clarification of the signaling cascade of apoptosis, their relationship with clinical manifestations during periodontal inflammation is almost completely unknown.
Hence, the aim of this study was to compare quantities of immunohistochemically identified p53, Bcl-2, and gingival tissue from patients with chronic periodontitis and gingivitis subjects.
To the best of our knowledge, limited studies have been conducted on comparing immunohistochemical components and special stains for the detection of apoptotic cells.
MATERIALS AND METHODS
Study Setting
The study was conducted at the Department of Periodontology and Oral Pathology & Microbiology, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Bengaluru, Karnataka, India.
Study Period
Subjects who reported to the Department of Periodontology, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Bengaluru, Karnataka, India, between December 2017 and March 2018.
So, each study group comprised of 10 samples. Individuals in the gingivitis group had mild signs of gingival inflammation, mild bleeding upon probing, a probing depth of <3 mm along with no clinical attachment loss. Patients in the periodontitis group had gingival inflammation, a probing depth of ≥5 mm, and clinical attachment loss greater than 3 mm. The excluded factors include patients with diabetes and other systemic illnesses, smokers, individuals on any kind of medication, and pregnant and lactating mothers.
Method of Specimen Collection
Subjects diagnosed with generalized periodontitis had their gingival tissue biopsied under local anesthesia. Biopsy sites were those where the probing depth was greater than 5 mm. Around 1–2 mm subgingival incisions were performed in inflammatory tissues. The biopsy specimens comprised of gingival epithelium and connective tissue.
The gingivitis samples were taken from individuals with gingivitis who had no periodontal disease. Tissues of patients seeking crown lengthening procedures and pericoronitis were considered.
The gingival tissue was harvested from the buccal or lingual site. Informed consent was obtained before the biopsy procedure and one sample was collected from each subject. The tissue was fixed in 4% neutral buffered formalin and transported to the laboratory where the specimens were routinely processed for histopathologic analyses. From the paraffin-embedded tissue, two sections of 4 μm thickness were taken on microslides, and one section each was stained using hematoxylin and eosin (H&E) and Feulgen reaction for DNA. Routine procedure for H&E staining was conceded. The procedure for Feulgen reaction for DNA staining is a specific method for selectively staining nucleic acids. Feulgen reaction is a semiquantitative technique. It is an aldehyde-specific reaction based on the formation of a purple-colored compound when aldehyde reacts with fuchsin-sulfuric acid—deoxyribonucleic acid gives this reaction after removal of its purine bases by acid hydrolysis; used as a nuclear stain.13 The staining intensity is proportional to the DNA concentration.
Visualization of apoptotic cells in the gingival epithelium was done under a light microscope at 100× magnification and was identified based on morphological features described by Kerr et al.14 Both H&E and Feulgen reaction for DNA staining sections were evaluated under light microscopy. Apoptotic cells were counted in 10 random fields. In each section, 1,000 cells were evaluated for the presence of apoptotic cells, and apoptotic index (AI) was calculated as the number of apoptotic cells expressed as a percentage of the total number of nonapoptotic cells counted in each case.15 AI of both sections was compared and statistical analysis was done using the Chi-square test, student t-test, Mann–Whitney test, and multivariant analysis.
For immunohistochemical staining, 4 µm-thick sections were deparaffinized and mounted on poly-L-lysine coated slides. The sections were placed in citrate buffer and heated in a microwave oven for 15–20 minutes at maximum power (700 W). This was then cooled at room temperature for 20 minutes. This was followed by incubation with primary antibodies for p53 (1:50; Dako Denmark) and Bcl-2 (1:50; Dako Denmark), diluted in 1% bovine serum albumin and phosphate buffered saline (PBS). This was incubated overnight at 4°C in a moist chamber. IHC staining was done with Envision system (HRP based two-step IHC staining method). Slides were then subjected to the chromogen, diaminobenzidine, followed by counterstaining with hematoxylin. Breast carcinoma (for p53) and tonsil tissue (for Bcl-2) were used as positive controls for immunostaining. Levels of expression of p53 and Bcl-2 on each slide were graded in a semiquantitative fashion using a scale of 0 to 3. + 0 = no staining; (1+) = stained cells comprising >10% of positive cells (2+) = stained cells comprising 30%, and (3+) = stained cells comprising >30% of positivity.
Statistical Analysis
The descriptive statistics included the calculation of mean, median values, standard deviation, and standard error of the mean for quantitative traits. For qualitative variables, a distribution of patient numbers in individual categories of a trait was analyzed. Statistical analysis took advantage of the independent student t-test for comparison of mean expression of apoptosis in gingival epithelium between two groups. Comparison between expression of apoptosis in gingival epithelium between two groups using multivariant analysis was done. Probability values <0.05 were considered significant.
RESULTS
Apoptotic cells of soft tissue gingiva exhibited well-differentiated cellular characteristics which include shrinkage of cell size, reduction of cytoplasm, and greater eosinophilic staining along with shrunk, circular to the crescent-shaped or haphazard-shaped nucleus. Cells undergoing apoptosis were seen to take up Feulgen nuclear reaction (FNR) stains deeply within the shrunk nuclei and dense FNR staining within the cytoplasm as well.
The apoptotic index was measured in a total of 20 cases including 12 males and eight females, which comprised 10 cases of gingivitis individuals (six males, four females) with a mean age of 27.1 years and 10 cases of chronic periodontitis (six males, four females) with a mean age of 44.8 years. Mean apoptotic index was higher in cases with chronic periodontitis as compared to that of gingivitis (Table 1).
Methods | Group | N | Mean | Standard deviation | Mean difference | t | p-value |
---|---|---|---|---|---|---|---|
H&E | Gingivitis | 10 | 4.74 | 3.01 | −8.31 | −6.518 | <0.001* |
Periodontitis | 10 | 13.05 | 2.68 | ||||
FNR | Gingivitis | 10 | 9.18 | 6.82 | −14.46 | −5.994 | <0.001* |
Periodontitis | 10 | 23.64 | 3.42 | ||||
p53 | Gingivitis | 10 | 15.54 | 12.48 | −16.20 | −3.500 | 0.003* |
Periodontitis | 10 | 31.74 | 7.65 | ||||
Bcl-2 | Gingivitis | 10 | 15.00 | 16.71 | 7.52 | 1.385 | 0.18 |
Periodontitis | 10 | 7.48 | 3.92 |
*indicates statistically significant
Apoptotic cells were more readily distinguishable in FNR-stained sections as compared with those that were stained by H&E, the difference was found to be statistically significant (p < 0.05) between the groups (Table 2 and Fig. 1). The cell organelles took a deeper stain as compared to the cytoplasm attributable to the higher cellular activity.
Variable | Group | Mean | Standard error | 95% confidence of interval | Mean difference | p-value | |
---|---|---|---|---|---|---|---|
Lower | Upper | ||||||
H&E | Gingivitis | 4.78 | 1.11 | 2.45 | 7.11 | −8.23 | <0.001* |
Periodontitis | 13.01 | 1.11 | 10.68 | 15.34 | |||
FNR | Gingivitis | 10.95 | 1.94 | 6.86 | 15.04 | −10.92 | <0.001* |
Periodontitis | 21.87 | 1.94 | 17.78 | 25.96 | |||
p53 | Gingivitis | 18.15 | 3.84 | 10.04 | 26.26 | −10.97 | 0.006* |
Periodontitis | 29.13 | 3.84 | 21.02 | 37.24 | |||
Bcl-2 | Gingivitis | 13.97 | 4.69 | 4.08 | 23.86 | 5.47 | 0.39 |
Periodontitis | 8.51 | 4.69 | −1.38 | 18.40 |
*indicates statistically significant
Fig. 1: Comparison of the mean expression of apoptosis in the gingival epithelium between two groups in different methods
Apoptotic cell count in the gingival epithelium was higher in the chronic periodontitis group with a mean AI of 23.64 compared to gingivitis where the mean AI was 9.18. The mean difference was found to be −14.46 and the difference was statistically significant (p < 0.001).
The immunohistochemical analysis revealed an elevated uptake of p53 indicating an increased immunoreactivity which was relatable to the increased inflammation of the periodontium. Here the apoptotic cell count in the gingival epithelium was higher in the chronic periodontitis group with a mean AI of 31.74 compared to the gingivitis group where the mean AI was 15.54. The mean difference was found to be −6.20 and the difference was statistically significant (p = 0.003).
Higher expression of antiapoptotic protein marker Bcl-2 was detected in the gingivitis group than in the chronic periodontitis group indicative of higher affinity of antiapoptotic marker Bcl-2 to cells with lower apoptotic activity or cells in better health compared to cells with higher apoptotic activity. Here the apoptotic cell count was higher in the gingivitis group with a mean AI of 15.00 compared to the chronic periodontitis group where the mean AI was 7.48. The mean difference was found to be −7.52 however the difference was not statistically significant (p = 0.18) (Fig. 2).
Figs 2A to F: (A) Feulgen-stained gingivitis section showing stratified squamous epithelium and inflammatory cells. No mitotic figures are observed (×100); (B) Feulgen-stained periodontitis section showing stratified squamous epithelium and inflammatory cells. Numerous mitotic figures are observed (×100); (C) IHC of gingivitis tissue showing low expression for antibody to p53, (×200); (D) IHC of periodontitis tissue showing high expression for antibody to p53 (×200); (E) IHC of gingivitis tissue showing high expression for antibody to Bcl-2 (×100); (F) IHC of periodontitis tissue showing mild expression for antibody to Bcl-2 (×100)
DISCUSSION
Periodontitis is a disease of multifactorial origin and involves continuous interplay between the defense mechanism of host and pathogenic bacteria, which ultimately leads to the damage of periodontium.16 Apoptosis is seen in physiological and pathological phenomena and constitutes a major role in homeostasis and morphogenesis. Apoptotic cells are found to be lower than other cells in the gingival tissue of chronic periodontitis.17
Programmed cell death or apoptosis has a significant part in the pathway of inflammation and the immune response combatted by the host. In the progression of apoptosis, a multitude of biochemical and morphological events take place in the cell which ultimately leads to cell death and subsequent removal by phagocytes. Apoptosis could hence be considered a significant process in the control of the host inflammatory reaction to combat chronic accumulation of bacteria and their products. Various catabolic proenzymes are already present within all living cells in a dormant form and they are processed to their active caspase catabolic enzyme in cells that enter the cycle of apoptosis.18,20
Gamonal et al. conducted a study on the apoptotic events of gingival tissue of adult patients with chronic periodontitis; a combination of both immunohistochemical staining and electron microscopy was employed to detect DNA fragmentation. The authors concluded that apoptosis is initiated within periodontal tissues because of both the microbial host and host components hence adding to the hypothesis that implies apoptotic phenomena in the inflammation cascade associated which involves gingival tissue destruction seen in periodontitis patients.21
The present study is an attempt to analyze the expression of apoptotic cells with H&E staining and assessment of apoptotic biomarkers p53 and Bcl-2 within the gingival epithelium.
In the present study, the apoptotic index across both stains showed a greater apoptotic index mean in chronic periodontitis samples as compared to gingivitis individuals, this finding was in agreement with the findings of a study by Aarati et al.14 who evaluated the apoptotic index among twenty cases, including ten cases each of chronic periodontitis and healthy samples. The mean AI was progressively increased in chronic periodontitis as compared to healthy gingiva in the above-mentioned study.
The family of Bcl proteins which is relevant to the apoptotic chain of events constitutes a few members that are capable of enhancing apoptosis (Bax, Bak, etc.) and also some proteins that can inhibit the phenomenon of apoptosis (Bcl-2, Bcl-xl). Haphazard regulation of the progression of apoptosis is often considered responsible for the initiation of autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus, and diseases of viral origin like acquired immune deficiency syndrome apart from the common bacterial infections.22 Several studies have demonstrated that attenuated bacterial toxins that were isolated from P. gingivalis could inhibit the apoptosis of neutrophils in low concentrations. This was attributed to the stimulated release of antiinflammatory cytokines by Bcl-2 present in these toxins.22
The present study showed a reduction in the intracellular uptake of Bcl-2 in the apoptotic cells, this finding was similar to the findings of a study by Gamanol et al., where there was a positive correlation associated with an increase in cytoplasmic levels of Bcl-2 in the neutrophils of periodontal tissues that exhibited a delay in apoptotic process in an animal model.22
The cellular protein p53 is often called a genome guardian, it plays a pivotal role in the repair of genetic repair of deoxyribonucleic acid, while if the repair of lesions is impossible, then it turns out to be an inducer of programmed death of the cell.5 It is also associated with the apoptosis of terminally differentiated cells, including cells of infiammatory infiltrate.6 Despite p53 also being present intracellular to normal cells and tissue, the rather short half-life of the protein makes its detection almost impossible in healthy tissue.23
The findings of the present study showed an elevation in the levels of p53 protein within the apoptotic cells of periodontitis cases as compared to the gingivitis patients, this finding was in conformance with a study conducted by Memmert et al. 2016 wherein the authors found that immunohistochemical analysis revealed an elevation of p53 immunoreactivity in accordance to the progression of periodontal inflammation.24
In the present study the p53 levels within the apoptotic cells of the gingival tissue were found to have a positive correlation with the extent of underlying inflammation, this finding was collinear to the findings of Kasprzak et al. 2014 where the authors found that there was higher expression of p53 in the gingival samples of patients with chronic periodontitis as compared to control group.25
CONCLUSION
Within the limitations of this study, it could be concluded that the apoptotic index was found to be higher in chronic periodontitis subjects as compared to subjects with gingivitis. p53 plays a pivotal role in periodontal ligament cell homeostasis and seems to be upregulated in oral inflammatory diseases. Bcl-2 being an antiapoptotic marker was associated more with gingivitis as compared to chronic periodontitis.
The apoptotic index was higher in chronic periodontitis as compared to gingivitis. p53 plays a pivotal role in periodontal ligament cell homeostasis and seems to be upregulated in oral inflammatory diseases. Bcl-2 being an antiapoptotic marker was associated more with gingivitis as compared to chronic periodontitis. In chronic periodontitis, significant alterations develop in the expression of immunocytochemical exponents of apoptosis, with the prevalence of Bcl-2 and p53 proteins, as compared to the control.
Further investigations necessitate studies with a larger sample size to find an association between chronic periodontitis and apoptotic biomarkers p53 and Bcl-2 in the gingival epithelium.
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