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B cell, Plasma cell, Oral lichen planus, Pathogenesis, IntroductionLichen planus is a chronic inflammatory disease, which affects skin, nail, oral and genital mucosa [ 1, ]. Oral lichen planus (OLP) has a prevalence of 0.1% to 4% in the population with a female predilection [ 2, ]. OLP is usually found in buccal mucosa, tongue, and gingiva [ 3, ]. Different clinical types of OLP include reticular, plaque-like, atrophic, erosive, and bullous pattern with the reticular form as the most common. Some investigators suggest that the plaque-like and erosive lesions have a potential for malignant transformation and development of squamous cell carcinoma [ 4, , 7, ]. The histopathological features include hydropic degeneration of basal cell layer, sub-epithelial band-like infiltration of lymphocytes, parakeratinized epithelium, keratinocytes apoptosis, and focal hyperparakeratosis of the epithelium [ 8, ]. The etiology of OLP is not completely understood, however, lymphocytic infiltration supports the hypothesis that OLP is a cell-mediated immune reaction or autoimmune reaction to keratinocytes. The characteristics of band-like lymphocytic infiltration are found to be a clue to etiopathogenesis of OLP [ 6, , 8, , 9, ]. Oral mucosa is exposed to variety of allergic materials such as dental restorative and casting materials like dental amalgam and Nickel. Foods like cinnamon and drugs are known to induce allergic reaction in the oral mucosa namely oral lichenoid reaction (OLR). Although OLR lesions are indistinguishable from OLP both clinically and histopathologically, unlike OLP, they do not go under malignant transformation. Moreover, contrasting OLP, lichenoid reactions secondary to dental restorative materials, medications, or foods can be resolved by changing restorative material or drug or food habits. &,zwnj Briefly, OLP and OLR are two distinct lesions with different causes that need different considerations [ 10, , 11, ]. Many studies have investigated the content of inflammatory infiltrate to distinguish OLR from OLP. Some researchers have attributed the presence of B-lymphocytes (B cell) and plasma cells in the lymphocytic infiltrate as one of the definitions of OLR lesions [ 12, , 14, ]. CD20 is a phosphor-protein expressed on B cells from pre B stage to the late stage of maturation. The expression decreases as B cells turn into plasma cells [ 15, ]. CD138 (syndecan-1) is a proteoglycan that facilitates cell-to-cell adhesion, cell and extra-cellular matrix interaction, cell differentiation and proliferation. CD138 is present on mature epithelial cells and plasma cells while is absent on endothelial and normal mesenchymal cells. Based on messenger RNA studies, CD138 is highly expressed on normal and neoplastic plasma cells and is absent on other cell types [ 16, , 18, ]. The aim of this study was to assess the presence of B cells and plasma cells in the inflammatory infiltrate of OLP by immunohistochemical (IHC) staining of CD20 and CD138 respectively. Moreover, the association between the presence of B cells and plasma cells with histopathologic features of the lesion was evaluated.Materials and MethodThis study cross-sectional study was conducted in Oral and Maxillofacial Department, Dentistry Faculty, Tehran University of Medical Sciences. It was ethically approved by Ethics Committee of Tehran University of Medical Sciences (ethical code, IR.TUMS.VCR.REC.1-395.1036).SamplesThe material in this study consisted of 61 biopsy specimens collected from the files of Oral and Maxillofacial Pathology Department, Tehran University of Medical Sciences from 2010 to 2017. The patients with the definite clinical and histopathological diagnosis of lichen planus with WHO criteria were included [ 19, ]. Patients with incomplete files, other autoimmune diseases including graft-versus-host disease (GVHD), lupus erythematous, history of medicine such as sulfonylurea, metformin, lorazepam and ketoconazole which are known to induce OLR [ 11, ], patients with single or unilateral lesions on buccal site and lesions that occurred in association with amalgam or glass ionomer tooth filings were excluded. Then the specimens, confirmed by an oral and maxillofacial pathologist, underwent IHC staining.The sections were reviewed by an oral and maxillofacial pathologist to determine the histopathologic features as follows, keratosis, acanthosis, granulosis, spongiosis, hydropic degeneration of basal layer, lymphocyte exocytosis, epithelium separation, intensity of inflammation (mild, moderate, and severe) and the degree of epithelial dysplasia (no, mild, moderate, severe and SCC) [ 20, ]. ImmunohistochemistryFormalin-fixed paraffin-embedded blocks were cut into 4&,micro m-thick sections and left 24 hours on silicone-coated slides. Afterwards, the sections were deparaffinized in xylene and immersed in methanol with 3% hydrogen peroxide for 5minutes to eliminate endogenous peroxide activity. Subsequently, the specimens were washed by citrated and left in microwave for 5min for antigen retrieval, then were cooled in room temperature for 30 min and washed by tap water for 10min. sections were treated with phosphate buffer saline (PBS) for 5 min and non-serum protein for 30min consecutively. Monoclonal antibodies were applied as follows for each section, CD20 (B cell, clone L26, Dako, Copenhagen, Denmark) and CD138 (clone CD138, Dako, Copenhagen , Denmark) diluted 1,200 and 1,100 respectively. They were left for 1 hour in room temperature, and then treated with PBS. Diaminobenzidine solution (Vector, Burlingame, CA, USA) 0.3% was used to visualize reaction products. As the last step, the sections were counterstained with Mayer&,rsquo s hematoxylin, dehydrated, and mounted. Normal tonsillar tissue was included as positive control for CD20. For CD138 internal control was used [ 9, ]. For negative control, sections were treated with normal saline and were confirmed to be unstained. Tonsillar tissue served as external control for CD20 and membranous staining of the oral epithelial cells served as the internal control for CD138.ScoringFor both markers, each section was examined by light microscope (OLYMPUS, BX51) at the magnitude of 200&,times in 10 randomly selected fields. The number of cells in the band-like inflammatory infiltration with membranous staining were counted and the percentage of stained cells were reported as grade 1 for no expression, grade 2 for less than 50% expression, and grade 3 for more than 50% expression [ 9, ]. Statistical analysisSPSS version 15.0 (SPSS Software Inc., CA, USA) was used to analyze the data. The correlation between CD20 and CD138 expression was assessed by Spearman correlation test. Fisher&,rsquo s exact test and chi-square test were employed to assess the difference of CD20 and CD138 expression in sections with different pathological features. The p&,lt 0.05 was considered statistically significant.ResultsOf the 61 specimens, 38(62.3%) belonged to female cases and 23(37.7%) belonged to male cases. The mean age of the patients was 49.6 years old. Buccal mucosa was the most frequent site of involvement (83.6%), and palate was the least frequent site with the frequency of 1.6 (Table 1,). Epithelial dysplasia was found in 15 cases (24.6%). CD20 immuno-expression was found in all cases (Figure 1,) while CD138 was expressed in 38 (62.3%) of cases (Figure 2,). The mean expression of CD20 and CD138 were found to be 22.5%&,plusmn 15.17% and 4.74%&,plusmn 9.23%, respectively (Table 2,). Min (%)Max (%)Mean (%)Expression by gradeCD20 expression28022.5&,plusmn 15.17Grade 10Grade 256(91.8%)Grade 35(8.2%)CD138 expression0414.74&,plusmn 9.23Grade 123(37.7%)Grade 238(62.3%)Grade 30Table 1.Expression of CD20 and CD138 in OLPs (Oral lichen planus)Figure 1. CD20 immuno-expression (&,times 200).Figure 2. CD138 immuno-expression in lymphocytic infiltration and epithelium as internal positive control (200&,times ).Number of specimensGrade of CD138 staining intensityp ValueGrade of CD20 staining intensityp ValueGrade 1Grade 2Grade 3Grade 1Grade 2Grade 3Type of lesion |