|Year : 2010 | Volume
| Issue : 3 | Page : 396-401
|E-cadherin and CD1a expression in gingival epithelium in periodontal health, disease and post-treatment
Ramya Arun, R Hemalatha, KV Arun, TSS Kumar
Department of Periodontics, Ragas Dental College and Hospital, Uthandi, Chennai, India
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|Date of Submission||28-May-2009|
|Date of Decision||01-Oct-2009|
|Date of Acceptance||23-Jan-2010|
|Date of Web Publication||29-Sep-2010|
| Abstract|| |
Background: Epithelial integrity is important for maintenance of periodontal health. It is not fully known if non-surgical periodontal therapy is capable of recreating the epithelial barrier in its functional state.
Patients and Methods: Sixty-five patients (31 males and 34 females) were included in the study. They were divided into group A (healthy gingiva 16 patients), group B (gingivitis 17 patients), group C (periodontitis 17 patients), and group D (post-treatment 15 patients). Gingival samples were collected and immunohistochemical study was done using E-cadherin and CD1a antibody. Statistical analysis was done using analysis of variance (ANOVA), followed by Tukey-Kramer multiple comparison test for CD1a and Tukey's highly significant difference (HSD) test for E-cadherin.
Result: There was a statistically significant difference (P<0.001) in the expression of E-cadherin between healthy (1.846±0.555), gingivitis (1.100±0.994), and periodontitis group (0.700±0.483). Similarly, there was a statistically significant difference (P<0.001) in the expression of CD1a between healthy (75.70±3.09), gingivitis (42.53±3.09), and periodontitis group (29.07±3.08). However, the expression of E-cadherin (1.242±0.653) and CD1a in post-treatment samples (52.18±2.90) was lower with no statistically significant difference when compared to health.
Discussion: The significant reduction in E-cadherin and CD1a levels in periodontal disease when compared to health could possibly be a result of invasion by the periodontopathogens and its subsequent sequel. Although, the post-treatment samples showed significant improvement when compared to disease, the reduction in E-cadherin and CD1a levels when compared to gingival health suggests that the epithelial barrier was not yet fully established in its functional state.
Keywords: CD1a, dendritic cells, E-cadherin, gingival epithelium, langerhans cells
|How to cite this article:|
Arun R, Hemalatha R, Arun K V, Kumar T. E-cadherin and CD1a expression in gingival epithelium in periodontal health, disease and post-treatment. Indian J Dent Res 2010;21:396-401
Gingival epithelium plays an important role in immunosurveillance of the periodontal tissues through the presence of immune cells and production of antimicrobial peptides.  Cellular component of the innate immune system present in the gingival epithelium includes cells such as dendritic cells and neutrophils.  Dendritic cells play a crucial role in antigen presentation to the T-lymphocytes and thereby help modulate the adaptive immune response to pathogenic stimuli. 
|How to cite this URL:|
Arun R, Hemalatha R, Arun K V, Kumar T. E-cadherin and CD1a expression in gingival epithelium in periodontal health, disease and post-treatment. Indian J Dent Res [serial online] 2010 [cited 2015 Mar 2];21:396-401. Available from: http://www.ijdr.in/text.asp?2010/21/3/396/70811
Dendritic cells have been classified into three major categories: two arising from the myeloid lineage and one from the lymphoid lineage. The dendritic cells of the myeloid lineage are Langerhans cells and the interstitial dendritic cells, also known as dermal dendritic cells.  Langerhans cells are dendritic cells present in the basal and suprabasal layers of the gingival epithelium. These cells act as the sentinels of the mucosa, whereby the immune system is directed to the presence of pathogens in the gingival epithelium. , There is some confusion in literature regarding the role played by these cells in periodontal disease. While some authors reported an increase in the number of Langerhans cells in gingival epithelium in periodontal disease,  others reported that Langerhans cells decreased in gingival epithelium in direct proportion to gingival inflammation. ,
In addition, the epithelium acts as a mechanical barrier through cell junctions such as E-cadherin. , E-cadherin is a calcium-dependant homophilic cell adhesion molecule that helps in cell-cell interaction. E-cadherin interaction is also thought to be important for retention of the Langerhans cells on the epithelial cells.  E-cadherin expression has been shown to be indirectly affected by periodontal disease, presumably as a result of the proteolytic destruction by putative periodontal pathogens such as Porphyromonas gingivalis. ,
Mechanical debridement continues to be an integral part of the periodontal treatment.  Significant improvement in clinical parameters such as bleeding on probing and pocket depth ensures that scaling and root planning is adequate as monotherapy in mild to moderate periodontitis.  Although external signs of inflammation exhibit significant improvement, there is still paucity in literature regarding the integrity of the epithelial barrier that is established after mechanotherapy.
A meaningful study of the gingival epithelium would require concomitant evaluation of both its mechanical and biological properties. The aim of the study, therefore, was to quantify E-cadherin and CD1a levels in gingival epithelium in periodontal health, disease, and after institution of phase I therapy.
| Patients and Methods|| |
Sixty-five subjects (31 males and 34 females) selected from the patient pool attending the outpatient clinic, Department of Periodontics, Ragas Dental College and Hospital, were enrolled in the study between January and June 2006. The Institutional Review Board of Ragas Dental College and Hospital approved the study protocol and informed consent was obtained from each subject. Subjects exhibiting good general health with no history of smoking or periodontal treatment or antimicrobial therapy for the past 6 months were chosen for the study.
The subjects were divided into four groups: group A consisted of 16 subjects (nine males and seven females; age: 22 to 34 years; mean, 30.7 years) with clinically healthy gingiva (probing pocket depth [PPD] <3 mm; no clinical attachment loss [CAL]; no bleeding on probing); group B consisted of 17 subjects (eight males and nine females; age: 31 to 41 years; mean, 35.5 years) with gingivitis (PPD 3 mm; no CAL; presence of bleeding on probing); group C consisted of 17 subjects (eight males and nine females; age: 36 to 45 years; mean, 39.5 years) with moderate periodontitis (PPD ≥5 mm and CAL ≤3 mm in at least six sites), and group D consisted of 15 subjects (six males and nine females; age: 36 to 45 years; mean, 39.5 years) who underwent phase I therapy and were re-evaluated after a period of 4 weeks when surgical periodontal therapy was undertaken due to persistent PPD of ≥5 mm. These sites, however, exhibited no signs of gingival inflammation.
Gingival samples were collected from subjects in group A while they underwent extraction for orthodontic reasons or during third molar removal; from subjects in group B during extraction of teeth for reasons other than periodontal pathology; from subjects in group C while they underwent extraction of teeth which had hopeless prognosis due to periodontal pathology and from subjects in group D during surgical periodontal therapy. The tissues obtained from the subjects were fixed with 10% neutral buffered formalin and then embedded in paraffin within 48 h to avoid antigen degradation. Sections that were 4 ΅m thick were made from the specimens and mounted on 3-aminopropyltriethoxysilane coated slides.
The tissues were deparaffinized by two changes in xylene (10 min each), put in descending grades of alcohol and then rehydrated with water. They were then transferred to citrate buffer, and antigen retrieval was done using microwave at power 4 for 30 min. Then the slides were dipped in two changes of phosphate buffered saline (PBS) for 5 min and then wiped carefully with gauze to remove excess PBS. The slides were treated with 3% hydrogen peroxide for 10 min, put in two changes of PBS, and then treated with power block for 20 min.
The primary antibody mouse monoclonal anti E-cadherin antibody (BioGenex) was added to the tissues and kept in room temperature for 1 h, after which the sections were washed in two changes of cold PBS (10 min each). After addition of super enhancer, incubation for 30 min and two changes of cold PBS, a drop of polyhorseradish peroxidase (HRP) was added onto the sections and incubated for another 30 min. Subsequently, sections were washed in two changes of cold PBS, and a drop of freshly prepared DAB (3'-diamino benzidine tetra hydrochloride - a substrate chromogen) was added. The slides were then washed in running distilled water and counter stained with Harris's Haematoxylin. After washing with acid alcohol and xylene, the tissue sections were mounted with disterene dibutyl phthalate (DPX) for microscopic examination at magnification 10x and 40x.
The tissues were deparaffinized using the procedure outlined above, transferred to tris-ethylene diamine tetra acetic acid (EDTA) buffer and autoclaved for antigen retrieval at 15 lbs pressure for 15 min. The slides were washed in two changes of distilled water and then dipped in tris buffer saline (TBS). The primary antibody mouse monoclonal anti CD1a antibody (Bio Genex) was added to the tissues and kept at room temperature for 1 h. The sections were then washed in three changes of cold TBS, a drop of biotinylated link was added and incubated 20 min. The slides were again washed in three changes of TBS; a drop of streptavidin was then added and slides incubated for 20 min. After immersing the slides in TBS for 5 min, a drop of freshly prepared DAB was added and the slides counterstained with Harris's hematoxylin. After washing with alcohol and xylene, the slides were mounted with DPX for microscopic examination at magnification, 10x and 40x.
E-cadherin expression was evaluated using the intensity of staining, whereas CD1a expression was evaluated by counting the number of cells that were stained.
E-cadherin: The expression of E-cadherin was scored as 0, 1, 2, and 3 depending on whether the intensity of staining was negative (-), mildly positive (+), moderately positive (++), or highly positive (+++), respectively.
CD1a: The percentage of positive cells was the ratio of the number of epithelial cells that showed intense staining divided by the total number of cells counted. A total of 1000 cells were counted in each slide. Thus, labeling index (LI)=(number of positive cells/1000)Χ100.
Data entry and descriptive analysis was performed using a statistical software program. Analysis of variance (ANOVA), followed by Tukey-Kramer multiple comparison test, was used to compare the groups with regard to CD1a antibody. ANOVA followed by Tukey's highly significant difference (HSD) test was used to compare the groups with regard to E-cadherin antibody.
| Results|| |
Mean intensity of staining of E-cadherin in the study groups
An intense brown membranous staining was observed in the basal and suprabasal layer of the epithelium in healthy gingival (group A), whereas no staining was observed in the connective tissue of gingiva. There was a decrease in the intensity of staining observed from samples in gingivitis (group B) and periodontitis groups (group C) with mild or no staining in samples in group C. The intensity of staining of post-treatment samples (group D) was greater than those in group B and C, but lesser than samples from group A [Figure 1](a-d). The mean intensity of staining [Table 1] was statistically significant (P<0.001) between group A (1.846±0.555); group B (1.100±0.994); and group C (0.700±0.483). However, there was no statistically significant difference between group D and group A (1.242±0.653).
|Figure 1 :Immunohistochemical expression of membrane staining E-cadherin (denoted by arrows) in (a) healthy gingiva (magnification 10x); (b) gingivitis group (magnification 40x); (c) periodontitis group (magnification 10x); and (d) post-treatment group (magnification 40x)|
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Mean labeling index of CD1a in the study groups
An intense cytoplasmic staining was observed in supra basal layers of the gingival epithelium in the healthy group(group A); whereas in the gingivitis (group B) and periodontitis (group C) group, the staining was milder. In the healthy group, the suprabasal cells showed increased reactivity when compared to basal portions. No staining was observed in the connective tissue [Figure 2]a-d.
|Figure 2 :Immunohistochemical staining for CD1a (denoted by arrows) in (a) healthy gingiva (magnification 10x); (b) gingivitis group (magnification 40x); (c) periodontitis group (magnification 40x); and (d) post-treatment group (magnification 10x)|
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The mean labeling index in individual groups were group A 75.70±3.09; group B 42.53±3.09; group C 29.07±3.08 and group D 52.18±2.90 [Table 2].There was a statistically significant difference between healthy gingiva and both gingivitis and periodontitis samples. There was also a significant difference between gingivitis and periodontitis groups at P-value are<0.001. However, there was no statistically significant difference between healthy gingiva and post-treatment samples (group D), although the post-treatment values were lesser than the health.
| Discussion|| |
An intact epithelium provides not only a mechanical barrier but also immunocompetent cells that regulate the immune responses of the periodontium.  Langerhans cells are an important component of the innate immune system that is present in the gingival epithelium. They are characterized by the presence of langerin, CD1a, and Birbeck granules. , In the present study, CD1a was used as a marker for Langerhans cells as it may be identified using immunohistochemical procedures fairly reliably. E-cadherin is the chief component of the adherens junction assembly in the gingival epithelium. E-cadherin is also responsible for assembling the proteins of the tight junction complex. , A high expression of E-cadherin in gingival health is, therefore, thought to correlate directly with the barrier properties of gingival epithelium.  The present study suggests that there was a decrease in CD1a expression in periodontal disease. These findings are in conformity with previously published reports. 
Although the role of dendritic cells has not been fully elucidated, several lines of evidence suggests a role for these cells in the pathogenesis of periodontal disease. Dermal dendritic cells can polarize effector responses toward Th1 or Th2, depending on the type of endotoxin or lipopolysaccharide used as a stimulant. Dermal dendritic cells have also been reported to increasingly infiltrate the lamina propria of gingiva in chronic adult periodontitis, and decrease after treatment.  Langerhans cells are believed to migrate into the connective tissue following antigenic stimulation such as that which occurs in periodontal disease.  The fate of these cells after they reach the connective tissue is not yet fully determined. While some authors suggests that they may migrate to the regional lymphnodes for T-cell activation,  others have reported formation of ectopic lymphoid organ within the gingiva.  Either way, their migration into the connective tissue provides an explanation for the decrease in Langerhans cell expression in gingival epithelium.
The decrease in E-cadherin expression observed in this study is also in agreement with previous studies. , The cysteine proteases (gingipains) secreted by Porphyromonas gingivalis been shown to be capable of proteolytic destruction of the α and β catenins that constitute the E-cadherin complex. , As E-cadherin is also important for Langerhans cell adhesion to keratinocytes,  the concomitant decrease in their expression in periodontal disease is along expected lines. Within the limitations of the study, it is difficult to establish if the reduced CD1a expression is a result of the decreased E-cadherin expression or if they have both reduced independently of each other.
The results of the present study suggest that there was a significant increase in E-cadherin and CD1a in post-treatment samples when compared to periodontal disease. However, these values are lesser, though not significantly so, when compared to health. These findings seem to suggest that the epithelium is not completely re-established to its pristine state of health. The mere absence of clinical signs of inflammation is thus not absolutely indicative of a return to a state of health, histologically. The post-treatment samples in the study were obtained only from those sites that exhibited PPD >5 mm without any external signs of inflammation. It is, thus doubtful, if these results can be extrapolated to all periodontal pockets.
The clinical implications of these findings cannot be fully explained on the basis of this study. These findings, however, warrant further investigation as re-establishment of epithelial barrier is a functional necessity for a healthy periodontium.
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Department of Periodontics, Ragas Dental College and Hospital, Uthandi, Chennai
[Figure 1], [Figure 2]
[Table 1], [Table 2]
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