Immunohistochemical evaluation of CD117 in mast cell of aggressive periodontitis.

CONTEXT: Aggressive periodontitis is a type of inflammatory response in periodontal tissues that is differentiated from chronic by its earlier onset, its high rate of progression, subgingival microbial composition, the difference in the host's immune response, and family history of the disease. Mast cells live in tissues and contribute to the various allergic and inflammatory conditions, including periodontal diseases, through the release of cytokines, chemokines, and proteolytic enzymes. CD117, the receptor of the stem cell factor expressed by the mast cells, is the hallmark of mast cell development. This study aimed to determine the expression level of CD117 in the mast cells of aggressive periodontitis using immunohistochemistry. AIMS: This study aimed to determine the expression level of CD117 in the mast cells of aggressive periodontitis using immunohistochemistry. SUBJECTS AND METHODS: The study was conducted on tissue samples from 15 patients with aggressive periodontitis and 15 patients with healthy gingival tissue. Samples were stained for CD117 markers using immunohistochemistry. After the microscopic examination of samples, all data were entered into SPSS 20 and analyzed. STATISTICAL ANALYSIS USED: Statistical data were analyzed using Kolmogorov-Smirnov test and independent t-test.
RESULTS: According to the results, the mean expression levels of CD117 in the aggressive periodontitis and normal gingival groups were 19.08 ± 6.9 and 18.2 ± 8.1, respectively, which were not statistically significant (P = 0.75). The Kolmogorov-Smirnov test verified the normality of distribution of CD117 expression level. Given the normal distribution of the scores, the independent t-test was used to compare the CD117 expression level in the two study groups.
CONCLUSIONS: There was no significant difference in the level of CD117 expression between the control and aggressive periodontitis groups. Copyright:

INTRODUCTION

Periodontitis is an inflammatory disease that is caused by the pathological response of the immune system to the bacterial activity. Aggressive periodontitis is a type of periodontal disease. Aggressive periodontitis has specific features such as rapid destruction of the bone supporting tooth and loss of attachment in young people, familial history of disease, and differences in the subgingival microbial composition and the host's immune response. Aggressive periodontitis can lead to an increase in pocket depth, a reduction in the periodontal supporting tissue, and ultimately loss of the tooth.[12]

The role of the immune system in the etiology and development of periodontal diseases has been confirmed, suggesting that the bacterial antigen can lead to an immunological response.[34] Mast cells originate from pluripotential hematopoietic cells found in bone marrow, where their various parts are differentiated. Subsequently, they enter the bloodstream and complete their differentiation in the connective tissue and peripheral mucosa.[567] Mast cells can secrete a range of inflammatory cytokines that have destructive effects on the periodontal tissue. Numerous studies have confirmed the role of mast cells in periodontal disease.[89] Mast cells contain secretory granules in which mediators are stored. Mediators will be released on the degranulation of mast cells. These mediators result in the destruction of the periodontal tissue.[8] The results of a study show that there is a relationship between the degree of mast cell degranulation and the severity of periodontal disease.[10]

CD117 (c-kit) is a tyrosine kinase type-3 protein; it acts as a receptor of the stem cell factor (SCF), which attaches to the SCF for the regulation of cell differentiation and proliferation, chemotaxis, and apoptosis.[11] FcεRI is a high-affinity receptor for immunoglobulin E (IgE). This receptor plays an important role in mast cell degranulation. Binding of SCF to the kit receptor results in enhanced FcεRI aggregation at the mast cell surface, leading to the degranulation of the mast cell. In other words, the c-kit ligand increased IgE-dependent degranulation.[121314]

CD117 can be useful for the detection of tumor and normal mast cells. CD117 is expressed in normal mast cells as well as mast cell tumors, and the expression of this marker in both is significant.[15] Fukuda et al. studied mast cell tumors and observed the expression of CD117 in the natural and humoral mast cells.[16] This study aimed to determine the expression level of CD117 in the mast cells presented in the aggressive periodontitis.

SUBJECTS AND METHODS

The study was conducted in accordance with the Helsinki Declaration and approved by the Institutional Ethics Committee (IR/ZAUMS/REC/1396-200). The purpose of the study was explained to the subjects and an informed written consent was obtained from the included patients and controls before the treatment. In this study, 15 samples of aggressive periodontitis and 15 samples of healthy gingival tissues were collected. Biopsy samples of aggressive periodontitis were from patients referred for surgery and healthy samples were from patients referred for clinical crown lengthening surgery at the Periodontics Department of the Faculty of Dentistry of the Medical Sciences University. The diagnosis of aggressive periodontitis was based on the radiographic analysis, medical history, familial history, and clinical findings.[17] The clinical findings are with regard to clinical attachment loss (CAL), bleeding on probing, and probing depths. In the radiograph, the amount and pattern of bone loss were evaluated for the diagnosis. In this study, samples were taken from a depth within the periodontal pocket. The site of the biopsy is shown in Table 1. In aggressive periodontitis group, 10 cases were generalized aggressive periodontitis and 5 cases were localized aggressive periodontitis. All patients had CAL and pocket depths more than 5 mm and had advanced aggressive periodontitis. The clinical findings of aggressive periodontitis patients are shown in Table 2. All patients were without systemic disease, were nonsmokers, and had no hormonal changes affecting periodontal conditions such as pregnancy, menstruation, or puberty. Patients did not take any medication that affects periodontal conditions. Samples were fixed in formalin 10%.

Table 1

Site of biopsy

Site of biopsy	Healthy, n (%)	Aggressive periodontitis, n (%)
Maxillary posterior	4 (26.6)	3 (20)
Maxillary anterior	3 (20)	1 (6.6)
Mandibular posterior	5 (33.3)	7 (46.6)
Mandibular anterior	3 (20)	4 (26.6)
Total	15	15

n – Number

Table 2

Mean of clinical finding in the aggressive periodontitis

	Number of CAL site of tooth involved	CAL (mm)	Number of tooth involved
Aggressive periodontitis	2.8	7.03	15.4

CAL – Clinical attachment loss; mm – Millimeter

Immunohistochemistry

All laboratory steps were carried out in a laboratory environment at 25°C in accordance with the BioGenex staining manual included in the kit.

To perform immunohistochemical (IHC) staining, the first 3-μm sections of the samples (gingival tissue) were prepared. The samples were then deparaffinized in xylene and hydrated using alcohol 80%, 90%, and 100%. The retrieval stage was performed using Tris buffer (pH = 7.6) for 30 min. Then, the jar containing the samples was indirectly cooled with running water. H2O2 (10%) was used for 10 min; then, the microscopic slide was washed with running water for 5 min. The samples were enclosed on the slide using a special pen.

The blocking serum was used for 5 min; then, the slide was shaken to remove the blocking serum. CD117 (BioGenex, The Hague, the Netherlands) initial antibody was used for 50 min; then, the slide was washed in a Tris-buffered saline (TBS) buffer. A secondary antibody was used for 1 h; then, the slide was washed again in a TBS buffer. Diammonium phosphate solution was added to the slide for 10 min and then washed in distilled water. Hematoxylin was used for 5 min and then washed again in distilled water. The slide was placed in alcohol 100 for 1 min and then placed in xylol for 2 min. Finally, the slides were mounted using a cytologic adhesive. Positive control samples, including gastric stomach tissue blocks, were also provided to ensure the accuracy of staining.

Histopathological evaluation

On completion of IHC staining, a Nikon optical microscope (Nikon, Type 4, Tokyo, Japan) with ×400 was used to count the number of mast cells at 10 high-power field in the highest density field (hot spot). The number of mast cells in each field was counted. Ten fields with the highest number of mast cells were selected, and the average number of mast cells in each group was recorded [Figures 1 and 2].

Figure 1

The mast cells contained in the normal gingival tissue specimen (immunohistochemical, ×400). (a) Mast cell

Figure 2

Mast cells in the aggressive periodontitis sample (immunohistochemical, ×400). (a) Mast cell

Statistical analysis

SPSS 20 software was used to analyze the data (SPSS Inc., Chicago, IL, USA). Normality of distribution of CD117 expression level was verified using the Kolmogorov–Smirnov test. Given the normal distribution of scores and percentages, the independent t-test was used to compare the expression of CD117 in the two study groups. The resulting P = 0.05 was considered statistically significant.

RESULTS

Clinical characteristics

The mean age in the aggressive periodontitis and the healthy group was 25.6 ± 2.5 and 37.3 ± 12.1, respectively. Twelve participants were male (40%) and 18 were female (60%).

Immunohistochemical findings

The mean expression level of CD117 in the aggressive periodontitis and healthy groups was 19.08 ± 6.9 and 18.2 ± 8.1, respectively, with no statistically significant difference (P = 0.75). Regarding the mean age difference in the two groups and the potential alteration of age variable, the difference between the two groups was also studied in terms of the expression level of CD117 in linear regression with the age variable control. There was no significant difference between the two groups in spite of the age variable control [Figure 3]. Furthermore, the level of CD117 expression without age variable control was evaluated, but the significant difference between the two groups was not observed [Table 3].

Figure 3

Box plot of the comparison of the CD117 expression level in the two groups expression of the CD 117 levels for control and aggressive groups (mean ± standard deviation)

Table 3

Expression level of CD117 in the aggressive periodontitis and healthy group

Variable	Mean±SD		Independent samples t-test (without age variable control)			Linear regression (with age variable control)P
	Aggressive group	Healthy group	t	df	P
Age	25.60±2.58	37.33±12.12	−3.66	28	0.001	0.733
CD117	19.08±6.91	18.21±8.16	0.313	28	0.756

P≤0.05 was considered statistically significant. SD – Standard deviation; df – Degrees of freedom; P – Probability value; t – Test statistic

DISCUSSION

Mast cells develop from myeloid progenitors by stimulating SCF. Activation of mast cells leads to degranulation and produces a range of pro-inflammatory substances that may lead to the onset of inflammation in the oral tissue. The gingival tissue in the oral cavity contains a large number of mast cells. Mast cells in periodontal disease were studied by a number of researchers and often different results were obtained because they used variable mast cell counting methods and the insufficient number of samples.[5918]

In this study, which examined the expression of CD117 markers in the mast cells of aggressive periodontitis, there was no significant difference between the healthy and the aggressive periodontitis groups. In the study by Gemmell et al., the number of c-kit-positive mast cells did not increase in periodontal disease,[5] which is similar to the result of our study. Furthermore, this study concluded that the Th2 response, which increases significantly in periodontitis, does not originate from mast cells.[5]

The results of other studies are also contradictory. For example, in some, they found that mast cell has a key role in the periodontitis disease, and in some others, there is no significant difference between aggressive periodontitis and healthy cases.[21019] A study by Malcolm et al.[20] concluded that there was a direct correlation between the reduction in the number of mast cells and the reduction of periodontal tissue degradation. According to another study, in dogs with inflammation treated with the lodoxamide ethyl (a mast cell degranulation inhibitor), the rate of bone loss was reduced compared with the untreated group. Therefore, control of mast cells leads to reduced periodontal degradation because factors that secrete mast cells directly lead to periodontal degeneration.[21] A study by Agrawal et al.[3] examined the relationship between the presence of mast cells and the different stages of periodontal disease. It showed that the number of mast cells in periodontal disease increased, indicating their presence and effect on the mechanism of periodontal disease. Therefore, it indicates the importance of these cells in the pathogenesis of periodontal disease and their role in the destruction of periodontal tissue.[3] Mast cells significantly express matrix metalloproteinase (MMP) and MMP is the key enzyme in soft periodontal tissue degeneration. This enzyme significantly expresses tumor necrosis factor which is a mediator that causes the spread of periodontal disease.[22]

In a new classification that was introduced by the American Academy of Periodontology and European Federation of Periodontology in 2017, chronic and aggressive periodontitis which was previously divided into two separate groups was combined into “periodontitis” category. In this classification, periodontitis was categorized based on grade (A, B, and C), stage (I, II, III, and IV), and extent of disease (localized and generalized). The stage is based on severity disease and complexity of disease management. The grade is based on the risk for rapid progression.[23]

According to the new classification,[23] due to the high rate of aggressive periodontitis progression, disease grade is considered as Grade C. According to the amount of CAL (at the site of greatest loss), bone, and tooth loss, the stage of disease is considered as Stages III and IV. Therefore, according to the level of CD117 expression, there was no significant difference between the control group and periodontitis with high grade and severe stage.

Because of the high prevalence of the periodontal disease, the lack of understanding of its mechanism, and the possible correlation with mast cells, further research is required with larger sample sizes and new methods to respond to appropriate therapies.

Limitations

The limitations of the study are considered the difficulty in finding a number of eligible samples in a limited time and failure to cooperate with a patient who needs to be encouraged to cooperate. Furthermore, in previous studies, there were limitations in choosing the method of counting mast cells and finding sufficient sample numbers.

CONCLUSIONS

The results suggest that there was no significant difference in the level of CD117 expression between aggressive periodontitis and healthy groups. Given the fact that few studies have been done in this field, more precise studies with larger sample sizes seem necessary.

In future studies, cell DNA examination and evaluation of specific mediators secreted from mast cells should be performed to achieve more conclusive and reliable results.

Financial support and sponsorship

This study was supported by the School of Dentistry, Zahedan University of Medical Sciences, Zahedan, Iran.

Conflicts of interest

There are no conflicts of interest.