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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 6  |  Issue : 1  |  Page : 30-35

Evaluation of the relation between T-Lymphocytes (CD4 + ve and CD8 + ve) and pemphigus vulgaris pathogenesis and severity


1 Department of Dermatology, Andrology and STDs, Faculty of Medicine, Mansoura University, Mansoura, Egypt
2 Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Date of Web Publication13-Jun-2019

Correspondence Address:
Dr. Mohammad A Gaballah
Department of Dermatology, Andrology and STDs, Faculty of Medicine, Mansoura University, El-Gomhoria St., Mansoura
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdpdd.ijdpdd_70_18

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  Abstract 


Background: Pemphigus vulgaris (PV) is an autoimmune bullous disease involving both the skin and mucus membranes. Infiltrating CD4+ and CD8+ T-lymphocytes may have a vital role in its pathogenesis. The autoimmune bullous skin disorder intensity score (ABSIS) was proposed for clinical evaluation and scoring of PV patients. Aims and Objectives: This work aimed to assess the role of CD4+ and CD8+ T-lymphocytes in the pathogenesis of PV and the correlation between number of T-lymphocytes infiltrate and clinical severity of the disease evaluated by ABSIS. Patients and Methods: Forty PV patients (patient group) and 10 healthy volunteers (control group) were enrolled. All patients were scored by ABSIS. Immunohistochemical staining was performed on skin biopsies for IgG, IgA, CD8, and CD4. Results: IgG was positive in 97.5% of patients, combined IgG and IgA were positive in 20% of patients, and only one case was negative for both antibodies. Thirty-three cases showed the predominance of CD4+ T cells, and seven cases showed predominance of CD8+ T cells. CD4/CD8 ratio was 2.578. Non-significant positive correlation was found between the total count of lymphocytic infiltrate and ABSIS. Conclusion: CD4+ and CD8+ T-lymphocytes share in the pathogenesis of PV and CD4+ are more expressed than CD8+ in the inflammatory infiltrate of PV lesions. The correlation between total lymphocytic count and ABSIS score may help to predict the clinical state of the patient from skin biopsy, and ABSIS score can predict the lymphocytic infiltration in PV cases. This correlation is in need to be reapplied on larger scales of patients to confirm its validity and reliability.

Keywords: Autoimmune bullous skin disorder intensity score, lymphocytes, pemphigus


How to cite this article:
El-Naby SM, Amin MM, Ali MA, Gaballah MA. Evaluation of the relation between T-Lymphocytes (CD4 + ve and CD8 + ve) and pemphigus vulgaris pathogenesis and severity. Indian J Dermatopathol Diagn Dermatol 2019;6:30-5

How to cite this URL:
El-Naby SM, Amin MM, Ali MA, Gaballah MA. Evaluation of the relation between T-Lymphocytes (CD4 + ve and CD8 + ve) and pemphigus vulgaris pathogenesis and severity. Indian J Dermatopathol Diagn Dermatol [serial online] 2019 [cited 2019 Nov 12];6:30-5. Available from: http://www.ijdpdd.com/text.asp?2019/6/1/30/260195




  Introduction Top


Pemphigus vulgaris (PV) is a severe potentially life-threatening organ-specific autoimmune bullous disease involving both the skin and mucus membranes and characterized by intraepithelial flaccid blisters.[1] It develops due to intercellular deposition of mainly IgG class antibodies against the extracellular domains of desmoglein 3 and to less extent desmoglein 1 leading to damage of desmosomes. Furthermore, antigen-antibody reaction activates the complement system resulting in further acantholysis, fluid accumulation, and characteristic vesiculobullous lesions.[2] The diagnosis of PV depends on a combination of clinical picture, histopathological findings, and diagnostic techniques for the detection of autoantibodies in the skin and blood.[3]

Direct immunofluorescence methods applied to specimens of perilesional skin are extremely reliable in demonstrating characteristic antibody deposition, and it is still the gold standard in differentiating vesiculobullous diseases.[4] However, this technique has its limitations that include the requirement for fresh tissue, impermanency of stained preparations, and the need for special equipment. Immunoperoxidase method largely overcomes these drawbacks due to its compatibility with fixed and paraffin-embedded tissue. In addition, it is sensitive and produces permanent staining that provides good morphological details which are clearly visible by light microscope.[5]

Infiltrating T-lymphocytes have a vital role in the induction and regulation of antibodies production in PV. Both autoreactive CD4+ve Th1 and Th2 cells may regulate the production of pathogenic autoantibody by B-lymphocytes.[6] Furthermore, CD8+ve T-cytotoxic cells were identified in PV, and they mediate immunity through regulation of B-cell functions, granzymes, or inducing apoptosis by the Fas/Fas ligand system.[7] The high density of T-lymphocytes in the bullous skin lesions and their perivascular location strongly suggests their important role in the development, exacerbation, and remission of PV.[8]

The autoimmune bullous skin disorder intensity score (ABSIS) was proposed by Pfütze et al.[9] on behalf of the German Blistering Disease Group for clinical evaluation and scoring of PV patients. The ABSIS has a total score ranging 0–206 (150 points for skin involvement, 11 points for oral involvement, and 45 points for subjective discomfit). It includes an objective component scored using body surface area and lesion type as weighting factors. The ABSIS also has a subjective component related to what the patient can eat, based on an oral disease scoring system for oral pemphigus proposed by Saraswat et al.[10] This component is scored according to the discomfort experienced during eating and drinking, with higher scores given to food with firmer textures such as fish or meat steak.

The aim of this work was to assess the role of CD4+ve and CD8+ve T-lymphocytes in the pathogenesis of PV and the correlation between these T-lymphocytes infiltrate and clinical severity of the disease.


  Patients and Methods Top


The present study comprised 40 patients diagnosed as PV attainted to the Dermatology outpatient clinic at Mansoura University Hospital from March 2015 to November 2015. Ten completely healthy volunteers were enrolled in this study as control group from who skin biopsy samples were taken from already removed extra skin during liposuction and skin tightening operation. Informed written consents were taken from all participants before participating in the study. This study was approved by the Institutional Research Board of the Faculty of Medicine, Mansoura University.

All participants were subjected to careful history taking, systematic general examination, and full dermatological examination. The clinical diagnosis of PV was according to the following criteria:[11]

  1. Multiple flaccid, thin-walled, easily ruptured vesicles, and/or bullae arise on either normal-appearing skin or erythematous bases
  2. Widespread cutaneous erosions with positive Nikolsky's sign (lateral pressure of unblistered skin at periphery of active lesions separates epidermis) and Asboe-Hansen sign (gentle pressure on intact bulla forces fluid to spread under skin away from the site of pressure)
  3. Shallow ill-defined irregular painful erosions and ulcers of different sizes of the oral mucosa.


All patients were subjected to clinical scoring by ABSIS.[9] Skin biopsies from patients and controls were taken. Patient with coagulation disorders, cardiac valve replacement, pregnancy, and tendency to keloid formation were excluded. Furthermore, too late lesions, ruptured, scratched, or traumatized lesions and lesions altered by topical or systemic treatment were avoided. Only intact blister with perilesional area of normal skin was biopsied. The skin tissue was put immediately in sufficient amount of 10% buffered formalin solution.

Histological analysis and immunohistochemistry:

  1. Sections of 4 μm thickness have been cut from formalin-fixed paraffin-embedded blocks of tissues for routine hematoxylin and eosin (H and E); others were prepared on charged slides for immunohistochemistry
  2. All slides were examined by H and E and examined using light microscope to diagnose PV and to evaluate inflammation
  3. The numbers of lymphocytes were counted in at least five random fields, and results were expressed as a mean per specimen. The areas of highest density of cells were chosen (hot areas)
  4. Immunohistochemical staining was performed for IgG (DAKO; ready to use, USA), IgA (DAKO; dilution range from 1:100 to 1:200, USA), CD8 (DAKO; ready to use, USA), CD4 (DAKO; ready to use, USA), on serial 4-μm sections using indirect avidin-biotin-peroxidase method. Positive and negative controls were done with each experiment
  5. Sections of PV cases were stained with anti-IgG antibody and anti-IgA antibody (for confirmation of diagnosis and exclusion of IgA pemphigus). IgG positive cases were stained with anti-CD4 antibody and anti-CD8 antibody to detect infiltrating T-cells density, distribution, and ratio
  6. For the quantitative study, stained cells were counted in several sites including dermoepidermal junction (DEJ), bullae, and perivascular areas; the average value was then calculated
  7. All images were analyzed using imageJ software to evaluate the immunopositive cells.


Data analysis

Data were entered and statistically analyzed using the Statistical Package for the Social Sciences (SPSS) version 20 software (IBM Corporation, Armonk, New York, USA). Qualitative data were described as numbers and percentages. Chi-square test was used for comparison between groups, as appropriate. Quantitative data were described as means (standard deviation) or medians, as appropriate. They were tested for normality by Kolmogorov–Smirnov test. In the nonnormally distributed variables, Kruskal–Wallis test and Mann–Whitney test were used for comparison between groups. P < 0.05 was considered to be statistically significant.


  Results Top


Demographic data, results of immunohistochemistry staining, median lymphocytic count, and ABSIS score in both patient and control groups were summarized in [Table 1] and [Figure 1], [Figure 2]. No cases showed IgA only as they were excluded from study.
Table 1: Demographic data, results of immunohistochemistry staining, total lymphocytic count, and Autoimmune Bullous Skin Disorder Intensity Score in both patients and control groups

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Figure 1: Pemphigus vulgaris (H and E, ×200)

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Figure 2: A case of pemphigus vulgaris shows positive intercellular stain for IgG (immunostain, ×200)

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The clinical evaluation of PV patients revealed blisters were found in 39 patients (97.5%), erosions and Nikolsky sign were found in 38 patients (95.0%), while crustations were found in 32 patients (80.0%). Combined skin and oral mucosal lesions were found in 33 patients (82.5%) while skin lesions alone were found in 7 patients (17.5%). The distribution of skin lesions showed that trunk was affected in 24 patients (60%), limbs lesions were found in 12 patients (30%), and generalized and scalp distribution each were found in 2 patients (5%). Oral affection distributed as follow: palatal lesions in 16 patients (48.5%), buccal lesions 15 patients (45.45%), and gingival lesions were found in 2 patients (6.06%).

The comparison between distribution of CD4+ve and CD8+ve T cells in skin sections showed that number CD4+ve T cells were significantly more than CD8+ve T cells in perivascular areas and the overall total distribution. Regarding the distribution of CD4+ve T-cells alone, they were significantly increased in perivascular than DEJ and bullae sites. Furthermore, CD8+ve T cells were significantly increased in perivascular than DEJ and bullae sites [Table 2] and [Figure 3], [Figure 4].
Table 2: Comparison between CD4+ and CD8+ in perivascular, dermoepidermal junction, bullae and total

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Figure 3: (a) A case of pemphigus vulgaris shows positive CD4 immunohistochemistry mainly in perivascular site (immunostain, ×100). (b) A case of pemphigus vulgaris shows positive CD4 immunohistochemistry mainly in perivascular site (immunostain, ×200)

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Figure 4: (a) A case of pemphigus vulgaris with positive CD8 immunohistochemistry mainly in perivascular site (immunostain, ×100). (b) A case of pemphigus vulgaris with positive CD8 immunohistochemistry (immunostain, ×100). (c) A case of pemphigus vulgaris with positive CD8 immunohistochemistry mainly in perivascular site (immunostain, ×200)

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The association between positive and negative CD4 patients, positive and negative CD8 patients, and ABSIS score and total lymphocytic count are summarized in [Table 3].
Table 3: Association between positive and negative CD4 cases, positive and negative CD8 cases, and Autoimmune Bullous Skin Disorder Intensity Score

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A statistically significant positive correlation was found between each of total CD4+ve T-cells count and total CD8+ve T-cells count and total lymphocytic count while no significant correlation was found between each of total CD4+ve T-cells count and total CD8+ve T-cells count and ABSIS score. There was statistically insignificant positive correlation between total lymphocytic count and ABSIS score (r = 0.245 and P = 0.114).


  Discussion Top


Immunoperoxidase was performed to all cases, and four features are used for the differential diagnosis of this test: immune deposition primary site; immunoglobulin class; immune deposits number; and other sites of immune deposition besides the main site. With these parameters, immunoperoxidase can lead to an accurate diagnosis in the majority of cases.[12]

In the current study, IgG deposition in the intercellular spaces was detected in 97.5% of PV patients while combined IgG and IgA positive patients were 20%. Habib et al.[13] reported positive IgG in 93% of patients, Ana Maria et al.[14] found positive IgG in 98% of patients, and Kabir et al.[3] detected positive IgG in 100% of PV patients. The discrepancy in the immunoperoxidase staining results may partly be due to poor selection of biopsy site and/or improper sampling technique in the referred cases. The older the lesion and the lesser the chances are for detecting autoantibodies due to repair mechanisms. Ana Maria et al.[14] reported some positive IgA cell debris inside the blister, on blood vessels in the upper dermis, and some individual cells in the upper dermis in some cases of PV in addition to IgG which was predominant in these cases.

The present study showed that the transition from the normal skin to the lesional skin (lesions with intraepidermal bullae) was associated with a significant increase in the density of total number of lymphocytes infiltrate in the lesional skin. These results are also detected in previous reports,[8],[15],[16] which may be due to increased lesional cells antigenicity, soluble factors (chemokines) production, increased adhesion molecule expression, and increased accessibility of tissue for immune cells mainly in perivascular location.

In the current study, the characterization of the infiltrate in PV perilesional skin was predominantly CD4+ T cells in 82.5% of patients, while 17.5% of patients showed predominantly CD8+ T cells. Both stains were statistically significant higher in perivascular location than DEJ and bullae, and this is in line with Giurdanella et al.[16] and Rizzo et al.[17] In addition, in the present work, CD4+ T cells were more expressed in PV skin lesions than CD8+ T cells with a ratio of 2.578. Giurdanella et al.[16] found that CD4:CD8 ratio was 2.7 with CD8 total cell count lower than the current study.

These results can confirm that the immunopathogenesis of PV involves both humoral and cell-mediated immune response. While antibodies to desmoglein are pathogenic, CD4+ and CD8+ T cells are also implicated. B-cell activation and antibody production classically necessitate the involvement of CD4+ T cells.[18] CD4+ Th1-cells promote the IgG1 and IgG2 production by B cells while IgG4, IgA, and IgE autoantibodies are induced by the cooperation of CD4+ Th2 with B cells. By contrast, CD8+ cells are responsive to in vitro stimulation with desmoglein 3, and they secrete interleukin 2 and interferon-gamma, being the first cytokines implicated in the cell-mediated immune response.[19] In addition, passive transfer of PV autoantibodies to CD8-deficient mice showed a lower incidence of pemphigus compared to the control group of wild-type mice. This suggests that CD8+ T cells may play a role in the pathogenesis of PV and development of acantholysis, perhaps through the granzymes or Fas/Fas ligand pathway that induces apoptosis.[16],[20]

In this study, ABSIS score was used for clinical evaluation of PV cases. The use of this score in clinical practice is valuable in the evaluation of PV patients, especially after the development of cutoff values of 17 and 53, to distinguish moderate, significant, and extensive pemphigus forms. ABSIS value of 17 points allows differentiation between moderate and significant pemphigus forms, and an ABSIS value of 53 points allows differentiation between significant and extensive pemphigus forms. Identifying these pemphigus subgroups should help physicians to classify and manage patients with pemphigus.[21] In addition, Hébert et al.[22] found that ABSIS score is a robust tool to accurately assess pemphigus activity.

The current study newly reported positive correlation between total lymphocytic count and ABSIS score which was not detected in previous reports. This correlation may help to predict the clinical state of the patient from histopathological evaluation of skin biopsy specimens and also add another advantage to ABSIS score that it can predict the lymphocytic infiltration in PV cases.


  Conclusion Top


CD4+ and CD8+ T-lymphocytes share in the pathogenesis of PV and CD4+ are more expressed than CD8+ in the inflammatory infiltrate of PV lesions. The correlation between total lymphocytic count and ABSIS score may help to predict the clinical state of the patient from histopathological evaluation of skin biopsy specimens and also add another advantage to ABSIS score that it can predict the lymphocytic infiltration in PV cases. This correlation is in need to be reapplied on larger scales of patients to confirm its validity and reliability.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Pan M, Liu X, Zheng J. The pathogenic role of autoantibodies in pemphigus vulgaris. Clin Exp Dermatol 2011;36:703-7.  Back to cited text no. 1
    
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Kabir AN, Das RK, Kamall M. Direct immunofluorescence test of skin biopsy samples – Results of 204 cases. Dinajpur Med Coll J 2009;2:8-12.  Back to cited text no. 3
    
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Schmidt E, Zillikens D. Modern diagnosis of autoimmune blistering skin diseases. Autoimmun Rev 2010;10:84-9.  Back to cited text no. 4
    
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Ratnatunga N, Ramadasa S, Doolwela Y. Diagnosis of pemphigus vulgaris with immunoperoxidase staining for IgG and C3. Ceylon J Med Sci 1992;35:11-3.  Back to cited text no. 5
    
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Veldman C, Feliciani C. Pemphigus: A complex T cell-dependent autoimmune disorder leading to acantholysis. Clin Rev Allergy Immunol 2008;34:313-20.  Back to cited text no. 6
    
7.
Hertl M, Veldman C. T-cellular autoimmunity against desmogleins in pemphigus, an autoantibody-mediated bullous disorder of the skin. Autoimmun Rev 2003;2:278-83.  Back to cited text no. 7
    
8.
Hussein MR, Ali FM, Omar AE. Immunohistological analysis of immune cells in blistering skin lesions. J Clin Pathol 2007;60:62-71.  Back to cited text no. 8
    
9.
Pfütze M, Niedermeier A, Hertl M, Eming R. Introducing a novel autoimmune bullous skin disorder intensity score (ABSIS) in pemphigus. Eur J Dermatol 2007;17:4-11.  Back to cited text no. 9
    
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Saraswat A, Bhushan K, India C. A new grading system for oral pemphigus. Int J Dermatol 2003;42:413-4.  Back to cited text no. 10
    
11.
Davenport S, Chen SY, Miller AS. Pemphigus vulgaris: Clinicopathologic review of 33 cases in the oral cavity. Int J Periodontics Restorative Dent 2001;21:85-90.  Back to cited text no. 11
    
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Cotell S, Robinson ND, Chan LS. Autoimmune blistering skin diseases. Am J Emerg Med 2000;18:288-99.  Back to cited text no. 12
    
13.
Habib R, Ashfaque AK, Syed AA. A study of Immunoperoxidase in the immunohistopathological diagnosis of autoimmune bullous diseases. J Pak Med Assoc 1988;38:154.  Back to cited text no. 13
    
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Ana Maria AV, Paul BG, Googe JR, Michael S. Immunohistochemistry versus immunofluorescence in the diagnosis of autoimmune blistering diseases. Our Dermatol Online 2013;4:585-95.  Back to cited text no. 14
    
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Caproni M, Giomi B, Cardinali C, Salvatore E, Pestelli E, D'Agata A, et al. Further support for a role for Th2-like cytokines in blister formation of pemphigus. Clin Immunol 2001;98:264-71.  Back to cited text no. 15
    
16.
Giurdanella F, Fania L, Gnarra M, Toto P, Di Rollo D, Sauder DN, et al. Apossible role for CD8+T lymphocytes in the cell-mediated pathogenesis of pemphigus vulgaris. Mediators Inflamm 2013;2013:764290.  Back to cited text no. 16
    
17.
Rizzo C, Fotino M, Zhang Y, Chow S, Spizuoco A, Sinha AA. Direct characterization of human T cells in pemphigus vulgaris reveals elevated autoantigen-specific Th2 activity in association with active disease. Clin Exp Dermatol 2005;30:535-40.  Back to cited text no. 17
    
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Pan M, Zhu H, Xu R. Immune cellular regulation on autoantibody production in pemphigus. J Dermatol 2015;42:11-7.  Back to cited text no. 18
    
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Takahashi H, Amagai M, Tanikawa A, Suzuki S, Ikeda Y, Nishikawa T, et al. Thelper type 2-biased natural killer cell phenotype in patients with pemphigus vulgaris. J Invest Dermatol 2007;127:324-30.  Back to cited text no. 19
    
20.
Grando SA, Bystryn JC, Chernyavsky AI, Frusić-Zlotkin M, Gniadecki R, Lotti R, et al. Apoptolysis: A novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis. Exp Dermatol 2009;18:764-70.  Back to cited text no. 20
    
21.
Boulard C, Duvert Lehembre S, Picard-Dahan C, Kern JS, Zambruno G, Feliciani C, et al. Calculation of cut-off values based on the autoimmune bullous skin disorder intensity score (ABSIS) and pemphigus disease area index (PDAI) pemphigus scoring systems for defining moderate, significant and extensive types of pemphigus. Br J Dermatol 2016;175:142-9.  Back to cited text no. 21
    
22.
Hébert V, Boulard C, Houivet E, Duvert Lehembre S, Borradori L, Della Torre R, et al. Large international validation of ABSIS and PDAI pemphigus severity scores. J Invest Dermatol 2019;139:31-7.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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