|Year : 2016 | Volume
| Issue : 2 | Page : 96-99
Oral epithelial cells in pemphigus vulgaris: An electronmicroscopic study in Indian population
Eesha Thakare1, Minal Chaudhary2, Madhuri Gawande2
1 Department of Oral Pathology and Microbiology, Nanded Rural Dental College and Research Center, Nanded, India
2 Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences, Wardha, Maharashtra, India
|Date of Web Publication||19-Dec-2016|
Shiv Multi Speciality Dental Clinic, First Floor, Akshay Plaza, Jatharpeth, Akola - 444 005, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim: Pemphigus, life-threatening illness affects only 1-5 patients per million populations per year. Cell junctions (desmosomes) are best visualized using either conventional or freeze-fracture electron microscopy. This is the first study in itself to document cellular morphologic details in patients of pemphigus vulgaris (PV) by scanning electron microscopy (SEM) in Indian population. This study was carried out with the aim of studying the cellular morphology and changes in the cell structures in patients of PV by SEM. Materials and Methods: The study consisted of four smears each from six patients belonging to the age group of 20-45 years comprising of two males and four females diagnosed for PV. The slides were then viewed under SEM. Results: The pathologic cell showed uneven cell boundaries with irregular arrangement of ridges forming a complex pattern; also the irregular elevations of plasma membrane formed a "plaque" like appearance on the cell boundaries. Conclusion: This study has added new observations related to the cellular changes and morphology seen in patients of PV, and numerous hypotheses are also stated to correlate with the possible etiopathogenesis of PV. In future, these observations may be useful in learning the pathogenesis of the disease even in the absence of frank lesions of the disease.
Keywords: Epithelial cells, microridges, pemphigus vulgaris, scanning electron microscopy
|How to cite this article:|
Thakare E, Chaudhary M, Gawande M. Oral epithelial cells in pemphigus vulgaris: An electronmicroscopic study in Indian population. Indian J Oral Health Res 2016;2:96-9
|How to cite this URL:|
Thakare E, Chaudhary M, Gawande M. Oral epithelial cells in pemphigus vulgaris: An electronmicroscopic study in Indian population. Indian J Oral Health Res [serial online] 2016 [cited 2020 Jul 11];2:96-9. Available from: http://www.ijohr.org/text.asp?2016/2/2/96/196122
| Introduction|| |
Pemphigus, life-threatening illness affects only 1-5 patients per million populations per year. Epidemiology of pemphigus has shown a different trend in India compared with Western literature in various counts.  The incidence of pemphigus among the dermatology outpatient attendees has varied widely, 0.09-1.8%.  The incidence assessed by clinic-based questionnaire survey conducted in Thrissur district, Kerala, was 4.4 per million population per year. The incidence was found to be higher than available data from Germany, France, and lower than Tunisia.  The Greek word "pemphigus" means a bubble or a blister. Different types of this disease are pemphigus vulgaris (PV), pemphigus foliaceous, pemphigus erythema, and pemphigus vegetans.  The peak incidence of PV occurs between the fourth and sixth decades of life with a male to female ratio of 1:2.  It consists of a group of epidermal diseases associated with bullae and acantholysis.  Specialized cell junctions occur at points of cell-cell and cell-matrix contact in all tissues, and they are particularly plentiful in epithelia. Cell junctions are best visualized using either conventional or freeze-fracture electron microscopy, which reveals that the interacting plasma membranes (and often the underlying cytoplasm and the intervening intercellular space as well). The importance of desmosomal junctions is demonstrated by some forms of the potentially fatal skin disease like pemphigus. Affected individuals make antibodies against one of their own desmosomal cadherin proteins. These antibodies bind to and disrupt the desmosomes that hold their skin epithelial cells (keratinocytes) together. This results in a severe blistering of the skin, with leakage of body fluids into the loosened epithelium. 
PV is an autoimmune skin disease characterized by intraepithelial blisters on the skin and mucous membranes and pathogenic antiepithelial autoantibodies which recognize the desmosomal glycoprotein desmoglein-3 (Dsg3) (1-3).  The term desmosome was later coined by Josef Schaffer in 1920 and is derived from the Greek words "desmo," meaning bond or fastening, and "soma," meaning body (Wells 2005; Calkins and Setzer 2007). The blisters in PV are located in the suprabasilar regions of the epidermis and are formed by a process of keratinocyte cell-cell detachment known as acantholysis  depending on the presence and distribution of Dsg3.
Extensive search of literature reveals few electron microscopic studies carried out on the PV. This is the first study in itself to document cellular morphologic details in patients of PV by scanning electron microscopy (SEM) in Indian population. Hence, the present study was carried out with the aim of studying the cellular morphology and changes in the cell structures in patients of PV by SEM.
| Materials and methods|| |
This study was conducted according to the institutional and ethical rules concerning research. Patients diagnosed clinicopathologically and through immunohistochemistry for Dsg3 for PV by Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital were considered for the study. The study consisted of four smears each from six patients diagnosed for PV. The patients belong to the age group of 20-45 years comprising of two males and four females. The physiologic cell population was taken from an apparently healthy person with no vesiculobullous disorder. Cytologic smears were obtained from the oral, buccal lesions of each patient by a wooden spatula. Specific glass slides of 1 cm × 1 cm were prepared, and smears were spread on these glass slides, fixed immediately in 2% glutaraldehyde for SEM analysis. The slides were then viewed under SEM microscope, VNIT Nagpur at X500, X2700, and X6000 respectively after electroplating and observations were made accordingly.
| Results|| |
The smears were viewed under SEM at X500, X2700, and X6000. A normal physiologic cell showed uniform cell boundaries at X500. At X2700 uniform arrangement of microridges with shallow vallies was evident. Nuclear space was evident. Regular architecture of plasma membrane with uniform arrangement of microridges was seen. The pathologic cell showed uneven cell boundaries. Few vallies were also appreciated on the cell surface. The irregular arrangement of ridges forming a complex pattern was observed [Figure 1]. At places, the irregular elevations of plasma membrane formed a "plaque" like appearance on the cell boundaries [Figure 2].
|Figure 1: Arrangements of microridges on the cellular surface of a cell in pemphigus vulgaris at different magnifications|
Click here to view
|Figure 2: The "plaque" like structure observed on the cellular surface of a cell in pemphigus vulgaris|
Click here to view
| Discussion|| |
Cellular evaluation of smeared oral cavity scrape samples plays a well-known role in the diagnosis of the majority of the oral lesions, including viral infection; because the method is a simple and inexpensive laboratory procedure. In this study, the pathologic cell showed uneven cell boundaries with few vallies on the cell surface along with irregular arrangement of ridges forming a complex pattern was observed. Our findings are in accordance with the findings of authors like Kobayashi et al. Cellular diagnosis introduced by Kobayashi et al.  is useful for the rapid demonstration of acantholytic epidermal cells in the bullae of PV. For this purpose, a smear is taken from the underside of the roof of the mouth and the base of the recently opened bulla. Although few investigations have applied exfoliative cytology to the diagnosis of pemphigus, they have noted that the cells are mostly single, but scraping yields a higher proportion of loosely attached cell clusters with prominent nucleoli and hyperchromatic nuclei.  Atypical acantholytic changes, which could often be misinterpreted as malignant cells, were sufficiently distinctive to avoid an erroneous diagnosis of a tumor. 
In regard to the surface topography of the oral cell samples, Kobayashi et al. first reported on the cell surface microfeatures in PV.  They suggested that cells change in their plasma membrane due to the destruction of their organelles and nuclei.  Their SEM appearance was similar to our cell picture, with stubby ridges. The topographic features of single acantholytic cells appeared somewhat different from those of the loosely attached cell clusters. There are numerous linear microridges with longitudinal elevations of the plasma membrane. Recognition of the acantholytic nature on SEM seem to depend primarily on the demonstration of the cell changes in their plasma membrane due to destruction of their organelles and nuclei.  It might be due to the dissolution of desmosomes provokes alteration, including dystrophy in microvilli.  Our observations [Table 1] and [Table 2] are in accordance to these findings. At places, the irregular elevations of plasma membrane forms a "plaque" like appearance on the cell boundaries [Figure 2]. This typical finding has not been reported in the previously reported cases on PV. A bundle of keratin intermediate filaments is attached to the surface of each plaque.  Transmembrane adhesion proteins of the cadherin family bind to the plaques and interact through their extracellular domains to hold the adjacent membranes together by a Ca 2+ -dependent mechanism.  However, since SEM is generally limited to the examination of surface morphology, demonstration of other diagnostic morphologic features, such as cell-to-cell adhesion changes (e.g., desmosome junction) is not possible.
|Table 1: Observations regarding morphology of normal physiologic cell under scanning electron microscopy|
Click here to view
|Table 2: Observations regarding morphology of pathologic cells of pemphigus vulgaris under scanning electron microscopy|
Click here to view
Over the past 10 years, progress in molecular biologic research has evolved gradually. Pemphigus is a class of diseases in which autoantibodies target the desmosomal cadherins, predominantly Dsg1 and Dsg3.  Although great strides have been made in identifying the pemphigus antigens, it remains unclear exactly how pemphigus antibodies cause loss of adhesion. One likely explanation is that antibody binding to the Dsg extracellular domain simply blocks adhesion by steric hindrance. , However, this hypothesis is called into question by several observations. For example, PV IgG is unable to induce acantholysis in plakoglobin-null cells. , In addition, human keratinocytes treated with PV IgG at 4°C do not show loss of cell-cell adhesion until the cells are shifted to 37°C, suggesting that keratinocyte responses are required in order for the antibodies to cause loss of adhesion.  Other studies have implicated a role for signal transduction in mediating PV-induced acantholysis. 
Pemphigus IgG binding has been shown to cause activation of numerous cell signaling pathways. Work by the Rubenstein laboratory has illustrated that PV IgG binding induced phosphorylation of heat shock protein 27 via p38 mitogen-activating protein kinase (p38MAPK). ,, p38MAPK has also been shown to be involved in phosphorylation of Dsg3 in response to PV IgG.  Furthermore, inhibition of p38MAPK activity prevents keratin retraction, actin reorganization and formation of epidermal blisters in a mouse model.  Similarly, signaling through the c-Myc pathway has also been implicated in PV pathogenesis.  These studies suggest that either the activation of signaling pathways on autoantibody binding to the desmosomal cadherins causes loss of adhesion, or alternatively, that manipulation of intracellular signaling pathways can bolster baseline adhesion strength and prevent blistering caused by pemphigus IgG.
A number of observations have also illustrated that pemphigus IgG binding may interfere with the normal turnover of the Dsgs. Early studies found that PV IgG was internalized after binding to the surface of keratinocytes. ,, Experiments have shown that PV IgG binding results in clathrin-independent endocytosis of Dsg3 and subsequent routing of the cadherin to a lysosomal compartment for degradation. , Furthermore, blocking Dsg3 endocytosis or upregulating Dsg3 biosynthesis by exogenously expressing Dsg3 prevents keratinocytic loss of adhesion in response to PV IgG. ,
Work performed by Payne and Kitajima also lends support to the impact of PV IgG on desmosomal assembly, as PV IgG causes internalization of newly synthesized pools of Dsg3. , Altogether, these findings suggest that pemphigus IgG disrupts the normal turnover and assembly of desmosomes. In fact, a growing body of evidence suggests that regulation of post-Golgi trafficking of cadherins is a key mechanism by which cell adhesion is regulated during development and disease. , New evolution with newer techniques will throw more light on the mechanism of etiopathogenesis of PV.
Few other diseases associated with loss of desmosomes or loss of Dsg4 are associated with defective hair follicle differentiation,  whereas Dsg1 haploinsufficiency leads to striate palmoplantar keratoderma, an epidermal thickening disease.  The localized impact of these mutations in Dsg1 and Dsg4 on the skin is consistent with the tissue expression patterns of these genes.  In contrast, mutations in Dsg2 result in arrhythmogenic right ventricular cardiomyopathy. ,
This study has added new observations regarding the cellular changes and morphology seen in patients of PV. This is the first study in itself to document cellular morphologic details by SEM. Numerous hypotheses are also stated to correlate with the possible mechanism of etiopathogenesis of PV. In future, these observations may be useful in diagnosing early cases of PV even in the absence of frank lesions of this dreadful disease.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Kanwar AJ, De D. Pemphigus in India. Indian J Dermatol Venereol Leprol 2011;77:439-49.
Shamim T, Varghese VI, Shameena PM, Sudha S. Pemphigus vulgaris in oral cavity: Clinical analysis of 71 cases. Med Oral Patol Oral Cir Bucal 2008;13:E622-6.
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 4 th
ed. New York, London: Garland Science; 2002.
Lin MS, Swartz SJ, Lopez A, Ding X, Fernandez-Vina MA, Stastny P, et al.
Development and characterization of desmoglein-3 specific T cells from patients with pemphigus vulgaris. J Clin Invest 1997;99:31-40.
Delva E, Tucker DK, Kowalczyk AP. The desmosome. Cold Spring Harb Perspect Biol 2009;1:a002543.
Kobayashi TK, Kaneko C, Sugishima S, Kusukawa J, Kameyama T. Scrape cytology of oral pemphigus. Report of a case with immunocytochemistry and light, scanning electron and transmission electron microscopy. Acta Cytol 1999;43:289-94.
Caldelari R, de Bruin A, Baumann D, Suter MM, Bierkamp C, Balmer V, et al.
A central role for the armadillo protein plakoglobin in the autoimmune disease pemphigus vulgaris. J Cell Biol 2001;153:823-34.
Calkins CC, Setzer SV, Jennings JM, Summers S, Tsunoda K, Amagai M, et al.
Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies. J Biol Chem 2006;281:7623-34.
Berkowitz P, Hu P, Warren S, Liu Z, Diaz LA, Rubenstein DS. P38MAPK inhibition prevents disease in pemphigus vulgaris mice. Proc Natl Acad Sci U S A 2006;103:12855-60.
Berkowitz P, Diaz LA, Hall RP, Rubenstein DS. Induction of p38MAPK and HSP27 phosphorylation in pemphigus patient skin. J Invest Dermatol 2008;128:738-40.
Berkowitz P, Chua M, Liu Z, Diaz LA, Rubenstein DS. Autoantibodies in the autoimmune disease pemphigus foliaceus induce blistering via p38 mitogen-activated protein kinase-dependent signaling in the skin. Am J Pathol 2008;173:1628-36.
Kawasaki Y, Aoyama Y, Tsunoda K, Amagai M, Kitajima Y. Pathogenic monoclonal antibody against desmoglein 3 augments desmoglein 3 and p38 MAPK phosphorylation in human squamous carcinoma cell line. Autoimmunity 2006;39:587-90.
Patel HP, Diaz LA, Anhalt GJ, Labib RS, Takahashi Y. Demonstration of pemphigus antibodies on the cell surface of murine epidermal cell monolayers and their internalization. J Invest Dermatol 1984;83:409-15.
Iwatsuki K, Takigawa M, Imaizumi S, Yamada M. In vivo
binding site of pemphigus vulgaris antibodies and their fate during acantholysis. J Am Acad Dermatol 1989;20:578-82.
Sato M, Aoyama Y, Kitajima Y. Assembly pathway of desmoglein 3 to desmosomes and its perturbation by pemphigus vulgaris-IgG in cultured keratinocytes, as revealed by time-lapsed labeling immunoelectron microscopy. Lab Invest 2000;80:1583-92.
Delva E, Jennings JM, Calkins CC, Kottke MD, Faundez V, Kowalczyk AP. Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism. J Biol Chem 2008;283:18303-13.
Mao X, Choi EJ, Payne AS. Disruption of desmosome assembly by monovalent human pemphigus vulgaris monoclonal antibodies. J Invest Dermatol 2009;129:908-18.
Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: An emerging feature of cancer. Nat Rev Cancer 2008;8:835-50.
Delva E, Kowalczyk AP. Regulation of cadherin trafficking. Traffic 2009;10:259-67.
Kljuic A, Bazzi H, Sundberg JP, Martinez-Mir A, O′Shaughnessy R, Mahoney MG, et al.
Desmoglein 4 in hair follicle differentiation and epidermal adhesion: Evidence from inherited hypotrichosis and acquired pemphigus vulgaris. Cell 2003;113:249-60.
Rickman L, Simrak D, Stevens HP. N-terminal deletion in a desmosomal cadherin causes the autosomal dominant skin disease striate palmoplantar keratoderma. Hum Mol Genet 1999;8:971-6.
Awad MM, Dalal D, Cho E, Amat-Alarcon N, James C, Tichnell C, et al.
DSG2 mutations contribute to arrhythmogenic right ventricular dysplasia/cardiomyopathy. Am J Hum Genet 2006;79:136-42.
[Figure 1], [Figure 2]
[Table 1], [Table 2]