Immunohistochemical Expression of HSP47 and CD206 in GCF and Fibroma of Oral Mucosa

Zahraa Naji Shenbooj^* and Ban F Al-Drobi

^*Correspondence: Zahraa Naji Shenbooj, Department of Oral Diagnosis, College of Dentistry, University of Baghdad, Iraq, Email:

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Introduction

The GCF is a mucosal fibrous mass that differs from other oral fibrous hyperplasia in a variety of ways. Its cause is yet unknown. It currently accounts for up to 5% of all oral fibrous proliferations submitted for biopsy. It did not seem to be caused by chronic irritation [1]. The GCF was first presented as a separate entity of fibrous hyperplastic soft tissue lesions by Weathers and Callihan in 1974. It got its name from its large, stellate-shaped, mononuclear, and multinucleated giant cells [2]. The GCF had no gender predilection [3,4]. The GCF has a little female predominance [1,5,6] and however, slight male predilection is reported in review of twenty-one GCF cases in Clinocopathological study of Sivaramakrishnan et al. [7]. Though GCF can affect any age, but it is most common in the first three decades of life [8]. Clinically, it appears as an asymptomatic raised lesion with a boss elated or pebbly surface, usually one centimeter or less in diameter. It may also be found in places other than the gingiva, such as the tongue, palate, and buccal mucosa. Unless traumatized during mastication or oral hygiene procedures, it typically has a normal mucosal color [4].

Histopathological, the GCF is covered by a thin layer of stratified squamous epithelium that is either parakeratinized or orthokeratinized, consists almost entirely of avascular moderately dense fibrous connective tissue with a fibromyxomatous stroma present in certain areas. Surface epithelium has elongated, small, and sometimes pointed rete processes, which are often accompanied by surface papules or boss elations, the existence of large giant cells usually with mono or bi-nuclei is considered as a consistent and diagnostic feature [9]. A number of authors have questioned whether the GCF should be classified as a distinct entity from the fibroma. They reached their conclusions based on the presence of the giant cells at different stages of the lesion's maturation, as well as the fact that other histological features aren't distinctive to support recognition as a separate entity [3,8,10].

Materials and Methods

Thirty cases of GCF and fibroma were retrospectively retrieved as formalin fixed-paraffin embedded tissue blocks (excisional biopsies). Tissue sections (5μm) were cut, mounted on positively charged slides, and immunohistochemically stained with monoclonal antibodies to HSP47 (ab109117, 1:300), CD206 (ab64693, 1:1000) using EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC kit (Abcam®, ab236466; 15ml).

The presence of a brown granular DAB staining pattern within the particular tissue compartment for a specific antibody in positive control tissue sections, but not in negative control tissue slides, was used to demonstrate Immunohistochemical signal specificity, according to manufacturer's datasheets.

All primary antibodies were observed and scored microscopically with a 400X objective in 5 representative fields for each tissue section; the average percent of the 5 high power fields was determined for each marker. All of the instances were evaluated blindly, with no prior knowledge of the other variables.

The Immunohistochemical staining for HSP47, CD206 antibodies was measured semi-quantitatively and assigned into categories for each one, as follows:

Hsp47 scoring: –, none, +, <10%, ++; 10-25%, +++, 26– 70%, ++++, 71% [11].

CD206 scoring: 0, none; 1, less than 5%; 2, 5-25%; 3, 25- 50%; and 4, more than 50% [12].

Results

Immunohistochemical findings

HSP47 expression is seen as brown cytoplasmic staining of stromal spindle and multinucleated giant cells in Figure 1. The distribution of study sample by HSP47 score, in GCF group 8 cases (53.3%) are scored four and 6 cases (40%) are scored three. In fibroma group, 10 cases (66.7%) are scored three and 3 (20%) are scored four (Figure 2).

Figure 1. HSP47 in GCF.

Figure 2. HSP47 expression in fibroma.

The comparison in mean percentage of HSP47 between GCF group and fibroma group revealed that there is a statistically significant difference in mean percentage of HSP47 between the studied groups. Mean percentage of HSP47 is significantly higher in GCF group compared with fibroma group (69.4% versus 53.2%, P=0.015) (Table 1).

Table 1: Comparison in mean percentage of HSP47 between the studied groups.

CD206 expression is seen as brown cytoplasmic staining of stromal spindle and multinucleated giant cells in Figure 3. The distribution of study sample by CD206 score revealed that the highest proportion of patients in both groups were with score three of CD206, 11 (73.3%) of GCF group and 8 (53.3%) of fibroma group (Figure 4). In this study the GCF group has significantly higher mean percentage of CD206 compared with those in fibroma group (67.5% vs. 55.2%, P=0.023) (Table 2).

Figure 3. CD206 expression in GCF.

Figure 4. CD206 expression in fibroma.

Table 2: Comparison in mean levels certain biochemical markers between study groups.

Correlations among immunohisto-chemical markers. In this study, we found a significant moderate positive correlation between percentage of HSP47 and percentage of CD206 of study groups (r=0.493, P=0.006) (Table 3).

Table 3: Correlation between HSP47 percentage and CD206 percentage.

Discussion

The GCF is a mucosal fibrous mass with many special characteristics that distinguish it from other oral fibrous hyperplasias [1]. HSP47, a collagen-specific molecular chaperone with a mass of 47 kDa, is found in the endoplasmic reticulum. During the folding, assembly, and transport of procollagen from the endoplasmic reticulum, HSP47 interacts with it transiently [13]. CD206 is a 175 kDa type I transmembrane glycoprotein that interacts with glycoproteins and collagen ligands and internalizes them. CD206 is expressed by cardiac resident macrophages, peritoneal macrophages, adipose tissue macrophages [14], placental macrophages, and skin macrophages [15].

Weathers and Callihan (1974) speculated that the giant cells could be melanocytes or Langerhans cells when they first reported GCF. In 1982, Houston added his supported the theory of weather and callihan. Several scientists' negative staining for S-100 [16-18] ruled this theory out. The presence of alpha-smooth muscle actin in the cells ruled out myofibroblastic origin. Negativity for CD68, LCA, and HLA-DR [18] overrides the macrophagemonocyte lineage.

The most accepted hypothesis is the fibroblastic lineage of giant cells that investigated by many authors and most of these authorities suggest the fibrous nature of GCF by 100% positivity of vimentin in their series, accordingly they stated that the characteristic giant cells are a giant fibroblast, the present study is the first study that used the HSP47 in GCF, it revealed an intense positivity of HSP47 in cases of GCF in comparison to fibroma which is in agreement with all previous Immunohistochemical studies that used the vimentin as a marker of fibrous differentiation [16,17,19-22], the comparison in mean percentage of HSP47 between GCF group and fibroma group revealed that there is a statistically significant difference in mean percentage of HSP47 between the studied groups. Mean percentage of HSP47 is significantly higher in GCF group compared with fibroma group (69.4% versus 53.2%, P=0.015). CD206, the mannose receptor, is a widely utilized macrophage marker. Although the CD206 is frequently used to recognize M2 macrophage subsets [23-26], its expression by other cell types (including satellite cells) has been documented [27].The present study is the first study in the evaluation of Immunohistochemical expression of CD206 in GCF lesions in which all cases of GCF were highly positive for CD206. In this study the GCF group has significantly higher mean percentage of CD206 compared with those in fibroma group.

As conclusions positive staining for HSP47 indicates that GCF has a mesenchymal differentiation that appears as spindle shaped cells forming the stromal tissue and mono-bi or multinucleated giant cells. Positive staining for CD206 in GCF and fibroma indicate that the marker is not specific for histiocytes differentiation and can stain other types of cells like fibrocyte so considered as fibrohistiocytic marker, and as a conclusion the GCF may considered to have a fibrous differentiation as the maker stain the fibrocyte cells in fibroma.

Ethical Approval

All experimental protocols were approved by the College of Dentistry, Dentistry, University of Baghdad. All experiments were carried out following the approved guidelines. (Ref no. 274 on 25/3/2021).

Financial Support

There was no financial disclosure.

Conflict of Interest

The authors declare that there are no conflicts of interest.

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