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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 2  |  Page : 646-653

Evaluation of hematopoietic stem cells in chronic discoid lupus erythematosus


1 Department of Dermatology, Faculty of Medicine, Menoufia University, Shibeen El Koom, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Shibeen El Koom, Egypt

Date of Submission13-Oct-2016
Date of Acceptance23-Nov-2016
Date of Web Publication27-Aug-2018

Correspondence Address:
Dalia Nassif
Medical Administration, Menoufia University, Shibeen El Koom 32817, Menoufia Governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.239734

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  Abstract 


Objective
The aim of the present study was to investigate the role of hematopoietic stem cell (HSC) and nonhematopoietic stem cells in lupus erythematosus through exploring the immunohistochemical expression of CD34 and c-kit.
Backgrounds
Chronic discoid lupus erythematosus (CDLE) is the most common type of cutaneous lupus erythematosus whose pathogenesis is still debated. The presence of autoantibodies in CDLE and the association between stem cell defects and autoimmunity encouraged us to investigate the possibility of HSC abnormality in CDLE through their immunohistochemical localization in skin samples of this disease using CD34 and c-kit antibodies.
Materials and methods
In total, 25 CDLE cases were selected, with 15 age-matched and sex-matched healthy individuals as a control group.
Results
CD34 showed positive expression in spindle-shaped dermal cells in 96% of CDLE cases with mild to strong intensity. CD34 was downregulated in CDLE cases compared with normal skin. Strong dermal intensity and higher Histo score (P < 0.001 for both) were significantly associated with normal skin. Regarding c-kit, spindle-shaped dermal cells showed positive immunoreactivity in 84% of cases, with mild to moderate intensity. Strong dermal intensity and higher dermal Histo score (P < 0.001 for both) were significantly associated with normal skin compared with CDLE cases.
Conclusion
We can conclude that HSCs are defective in CDLE cases as proved by CD34 and c-kit immunostaining. Further research is needed to investigate bone marrow HSCs and the underlying molecular mechanisms for such defect. Experimental studies using HSC transplantation for resistant or disseminated CDLE cases may be evaluated.

Keywords: chronic discoid lupus erythematosus, hematopoietic stem cells, immunohistochemistry, pathogenesis


How to cite this article:
Seleit I, Bakry OA, Samaka RM, Nassif D. Evaluation of hematopoietic stem cells in chronic discoid lupus erythematosus. Menoufia Med J 2018;31:646-53

How to cite this URL:
Seleit I, Bakry OA, Samaka RM, Nassif D. Evaluation of hematopoietic stem cells in chronic discoid lupus erythematosus. Menoufia Med J [serial online] 2018 [cited 2018 Dec 11];31:646-53. Available from: http://www.mmj.eg.net/text.asp?2018/31/2/646/239734




  Introduction Top


Lupus erythematosus (LE) is a chronic, connective tissue disorder that includes a broad spectrum of symptoms. LE follows a chronic course with sudden exacerbations and periods of remission. The cutaneous manifestations of LE can be divided into LE-specific and non-LE-specific skin manifestations based on histopathological findings [1]. The LE-specific skin manifestations show a typical histopathological picture with a lichenoid tissue reaction. They can be further subdivided into acute cutaneous LE, subacute cutaneous LE, and chronic cutaneous LE [2].

Chronic discoid lupus erythematosus (CDLE) is the most common subtype of chronic cutaneous LE. In 6080% of cases, lesions are localized above the neck, and in 2040%, lesions are generalized (both above and below the neck) [3]. Overall, 7090% of patients experience photosensitivity [4].

CDLE has multifactorial etiology. A genetic predisposition is essential. The disease is then affected by different environmental causes. Triggering factors include ultraviolet light, medications, smoking, hormones, stress, infections, and skin trauma [5].

Ultraviolet light is able to induce proinflammatory cytokines like tumor necrosis factor-α and interleukin-1 in both keratinocytes and lymphocytes. This leads to the induction of local inflammatory mediators like chemokines and lipid mediators, which amplify the subsequent inflammatory response at the local level [6].

In a study by Biazar et al. [7], anti-Ro/SSA and anti-LA/SSB antibodies were found in 22% and in 7% of patients with CDLE, respectively. One of the proposed mechanisms for the development of autoantibodies involves a defect in apoptosis that causes increased cell death and a disturbance in immune tolerance [8].

However, it still remains controversial, whether the tissue destruction in CDLE is owing to attack by the inflammatory cells of the mononuclear infiltrate or to antibody deposits at the dermal–epidermal junction or to other unknown mechanisms [9].

Stem cells are characterized by their capacity for self-renewal, multilineage differentiation, proliferation, mobilization, and homing [10]. Stem cells can be divided into two main groups: embryonic stem cells and adult stem cells [10].

The stem cells most commonly used in clinical medicine are hematopoietic stem cells (HSC), a type of multipotent adult stem cells, which give rise to all types of blood and immune system cells [10].

Autoimmune diseases are characterized by the breakdown of peripheral and central immune tolerance affecting immune processes, executed by autoreactive T and B cells, subsequently leading to tissue damage and functional loss [11].

Immune deregulation in autoimmune diseases may affect bone marrow (BM) progenitor cell development at several stages of differentiation and/or BM stromal cell function [12]. Autoreactive lymphocytes and proinflammatory mediators have been primarily implicated in BM damage, in that, these elements suppress the hematopoiesis-supporting capacity of BM stromal cells and accelerate the apoptosis of HSC/progenitor cells [13].

On the contrary, Ikehara [14], based on animal models, suggested that autoimmune diseases may be seen as primary stem cell disorders.

However, other reports suggested that patients with SLE have a defect in the BM microenvironment [15].

Therefore, HSCs are adversely affected in autoimmune disorders either primarily or secondary to stromal dysfunction.

The presence of autoantibodies in CDLE, the link between HSC defects and autoimmunity, and the ability of these cells to migrate from BM to other tissues encourage us to investigate the possibility of HSC abnormality in CDLE through their immunohistochemical localization in skin samples of this disease using CD34 and c-kit antibodies.


  Materials and Methods Top


Ethics

A written consent form approved by the Local Ethical Research Committee in Menoufia Faculty of Medicine was obtained from every individual before the study initiation. This was also in accordance with the Helsinki Declaration of 1975 (revised in 2000).

Patients

This case–control study included 40 individuals. These included 25 cases with CDLE and 15 age-matched and sex-matched healthy individuals as a control group. Cases were selected from Dermatology Outpatient Clinic, Menoufia University Hospital, during the period from March 2014 to March 2015.

Normal skin samples were obtained from individuals attending Plastic Surgery Department. Biopsies from cases and controls were site matched.

Clinical data describing patients' demographics (age and sex) as well as the clinical variables (site of lesions, disease duration, family history of CDLE, and the presence of nail, conjunctival, joint, or oral mucosa affection) were obtained and documented.

The diagnosis of CDLE was made based on patient history, clinical presentation, and clinical features that were confirmed by histopathological examination. Selected patients were either newly diagnosed or stopped medical treatment (topical and/or systemic) for at least 4 weeks before biopsy taking.

Skin biopsies

Three millimeter punch biopsies were taken under local anesthesia from cases and controls. Biopsies were fixed in neutral formalin 10% and subjected to routine tissue processing, ending with paraffin-embedded block formation at the Pathology Department, Faculty of Medicine, Menoufia University.

Serial sections, each 4 μm thick, were cut from each block; one was stained by hematoxylin and eosin to confirm the diagnosis and the other sections were cut on positively charged slides for the immunostaining procedure.

Histopathologic evaluation

Hematoxylin and eosin-stained sections were assessed to evaluate and verify epidermal and dermal pathological changes.

Immunohistochemical staining for CD34 and c-kit

The method used for immunostaining was streptavidin–biotin amplified system. The slides were submitted to subsequent steps of deparaffinization and rehydration. Antigen retrieval was done by boiling in citrate buffer saline (pH 6) followed by cooling at room temperature. The primary antibodies were incubated overnight at room temperature, and then the secondary antibody was applied with diaminobenzidine as a chromogen substrate and Mayer's hematoxylin as a counter stain. Primary antibodies were mouse monoclonal anti-c-Kit Antibody (clone T595; Novocastra, London, United Kingdom) and mouse monoclonal anti-CD34 antibody (clone QBEnd/10; Cell Marque, Rocklin, California, United States). Both antibodies were received as 1.0 ml concentrated and diluted by phosphate buffer saline in a dilution 1: 75.

The reaction was visualized by an appropriate substrate/chromogen (diaminobenzidine) reagent with Mayer's hematoxylin as a counter stain.

Interpretation of the immunostaining results

For each antibody, the epidermis and dermis, both in lesional and perilesional areas, were assessed.

Interpretation of c-kit immunostaining

Positive immunostaining was identified by brownish cytoplasmic or membranous discoloration [16].

Interpretation of CD34 immunostaining

Positive immunostaining of CD34 was identified by brownish cytoplasmic discoloration [17].

For both antibodies, the following items were evaluated:

  1. Staining intensity was assigned subjectively to be one of four grades: grade 0: no detectable staining, +: mild staining, ++: moderate staining, and +++: intense staining [18]
  2. Histo score (H score) was applied to evaluate the positive cases, where both the intensity and the percentage of positivity were considered using the following formula: H score=+3 (strong intensity)×% of + 2 (moderate intensity)×% of + 1 (mild intensity)×%. The score ranges from 0 to 300 [19].


Statistical analysis

Data were collected, tabulated, and statistically analyzed using a personal computer with SPSS version 11 program (SPSS Inc., Chicago, Illinois, USA). Values were expressed as number, percentage, and mean ± SD when appropriate. c2-test was used to study the association between two qualitative variables. Student's t-test was used for comparison between two groups having quantitative variables. The Pearson coefficient (r) was used to measure the correlation between the two quantitative variables. Differences were considered statistically significant with P less than 0.05.


  Results Top


Clinical data of selected cases are shown in [Table 1].
Table 1: Description of clinical data of studied discoid lupus erythematosus cases

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Immunohistochemical expression of CD34 in studied groups

Normal skin

CD34 was not expressed in the epidermis and hair follicles of all examined sections. Dermis showed strong staining of endothelial lining of resident blood vessels and in stromal cells in the vicinity of blood vessels, eccrine sweat glands, and pilosebaceous units in all sections. Dermal H score has mean ± SD of 190.67 ± 23.14 [Table 2] and [Figure 1].
Table 2: Comparison between CD34 expression in chronic discoid lupus erythematosus cases and control group

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Figure 1: (a) Control healthy skin showed absolute negativity of epidermis for CD34. Dermis showed CD34+ cells distributed periadnexally and diffusely in stroma (IHC, ×100). (b) Control skin showed CD34+ cells in dermis with perieccrine sweat gland ducts localization and accentuation (CD34) (IHC, ×400). IHC, immunohistochemistry.

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CDLE cases

CD34 was not expressed in the epidermis or hair follicles of all examined cases. Dermis showed positive expression in adnexa and spindle-shaped dermal cells in 96% of cases with mild to strong intensity. Dermal H score has mean ± SD value of 106.66 ± 27.77 [Table 2] and [Figure 2].
Figure 2: (a) A case of CDLE showed negative CD34 immunoreactivity in epidermis. Dermis showed positive moderate immunoreactivity for CD34 in spindle-shaped cells (IHC, ×40). (b) A case of CDLE showed positive moderate immunoreactivity of CD34 in interstitial stromal cells adjacent to eccrine ducts with built in control of small blood vessels (IHC, ×400). (c) A case of CDLE showed positive mild immunoreactivity of CD34 diffusely in spindle-shaped stromal cells (IHC ×400). CDLE, chronic discoid lupus erythematosus; IHC, immunohistochemistry.

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Comparison between CD34 expression in CDLE cases and control group

CD34 was downregulated in CDLE cases compared with normal skin. Strong dermal intensity and higher H score (P < 0.001 for both) were significantly associated with normal skin [Table 2].

Immunohistochemical expression of c-kit in studied groups

Normal skin

C-kit was expressed in the epidermis of all sections with moderate to strong intensity. It was expressed in hair follicles in 20% of cases in the inner and outer root sheaths. C-kit was expressed in the dermis of all sections (in spindle-shaped stromal cells) with moderate to strong intensity. Dermal H score has mean ± SD value of 182.7 ± 26.6 [Table 3] and [Figure 3].
Table 3: Comparison between cases and control group regarding c-kit expression

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Figure 3: A healthy skin showed moderate positive c-kit immunostaining in epidermis and hair follicles and mild in dermis (IHC, ×40). Inset photo is a higher power view of the same section (IHC, ×400). IHC, immunohistochemistry.

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CDLE cases

C-kit was expressed in the epidermis of 80% of cases, with mild to strong intensity. It was expressed in hair follicles in 24% of cases. Spindle-shaped dermal cells showed positive immunoreactivity in 84% of cases, with mild to moderate intensity. Dermal H score has mean ± SD value of 90.0 ± 32.7 [Table 3] and [Figure 4].
Figure 4: (a) A CDLE case showed negative c-kit expression in epidermis and dermis; however, hair follicles displayed mild positive c-kit expression (IHC, ×100). Inset photo showed high-power view of the same field (IHC, ×400). (b) A CDLE case showed strong positive c-kit expression in all layers of epidermis. Dermis showed positive c-kit expression in spindle-shaped stromal cells (IHC, ×40). CDLE, chronic discoid lupus erythematosus; IHC, immunohistochemistry.

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Comparison between cases and control group regarding c-kit expression

Strong epidermal intensity, higher epidermal H score, strong dermal intensity, and higher dermal H score (P < 0.001 for all) were significantly associated with normal skin compared with CDLE cases [Table 3].

Relationship between CD34 and c-kit expression and clinicopathologic data of studied cases

No significant association was detected between CD34 intensity or H score and clinicopathologic parameters of studied cases (data not shown in tables or figures).

Moderate intensity of c-kit expression was significantly associated with younger age of cases (data not shown in figures or tables).

Correlation between dermal CD34 H score with epidermal and dermal c-kit H score

There was a significant positive correlation between dermal CD34 H score and both epidermal and dermal c-kit H score [Figure 5].
Figure 5: Correlation between dermal CD34 H score with epidermal and dermal c-kit H score. (DE.CD.HS: dermal CD34 H score; DE.cytokines.HS: dermal c-kit H score; EP.CK.HS: epidermal c-kit H score). CD, chronic discoid; DE, dermal–epidermal; EP, epidermis; HS, hair shaft; H score, Histo score.

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  Discussion Top


In the present work, evaluating CD34 immunoreactivity in normal skin showed positive expression in the endothelial lining of resident blood vessels and in stromal cells near blood vessels, eccrine sweat glands, and pilosebaceous units. This was in agreement with previous reports [20].

The positive c-kit expression in epidermis of normal skin and within the hair follicle compartments went with earlier studies on human skin [20],[21]. C-kit protein plays some roles in the process of normal cellular regeneration in human keratinocytes [21] through its ability to promote reparative angiogenesis and to inhibit endothelial apoptosis [22].

Dermal CD34 was downregulated in CDLE compared with normal skin. The expression of cutaneous CD34+ cells was not investigated before in systemic or CDLE. However, there is evidence that patients with SLE present BM dysfunction, with a marked reduction in the numbers of BM CD34+ cells and a possibly reduced stem cell proliferation capability. The patients present elevated levels of apoptosis in the CD34+ cell subpopulation and a reduced frequency of colony-forming units when compared with control groups [16]. These findings have been further substantiated by studies demonstrating low numbers of peripheral blood CD34+ cells in association with increased apoptosis [23].

These changes were explained by increased expression of Fas antigen in these cells [13].

Fas and Fas ligand were positively expressed in most epidermal keratinocytes, infiltrating lymphocytes around blood vessels and appendages in CDLE skin and were upregulated compared with control healthy skin [24], but their expression was not evaluated in CD34+ cells.

The mechanisms leading to upregulation of Fas in CD34+ cells in patients with SLE are unknown. Normal BM CD34+ cells do not express Fas antigen on their surface, or they express the molecule at a proportion of 10% [25]. It has been reported that interferon-δ and tumor necrosis factor-α can induce the expression of functional Fas molecules on cultured normal hematopoietic progenitor cells [26].

Therefore, we can assume that the observed CD34 downregulation in our cohort of cases is because of decreased BM CD34+ and increased cell apoptosis leading to decreased immigrant cells to the skin. Future research on larger number of cases is mandatory to confirm or deny this suggestion. Evaluating BM CD34+ cells in CDLE cases and BM and tissue expression of Fas in these cells is also strongly recommended.

Dermal c-kit expression was downregulated in studied cases compared with healthy controls. C-kit expression was addressed before in SLE cases in BM HSCs. It was found to be upregulated, with no significant difference between cases and controls, in an attempt to enhance the growth and differentiation of hematopoietic cells [27]. However, it was not investigated in CDLE.

In the present work, strong intensity of CD34 and c-kit was significantly associated with younger age of patients. This finding went with Bakry et al.[20]. Krishnamurthy and Sharpless [28] mentioned that, as we grow older, our stem cells might lose replicative capacity. However, Sharpless and DePinho [29] stated that SC numbers and self-renewal capabilities do not necessarily decline with age, but rather its ability to produce progenitors and differentiated effector cells declines with age.

Significant positive correlation between dermal immunoreactivity of CD34 and c-kit in CDLE was demonstrated here. There was no comment on this observation before, but it was previously reported that within the human hematopoietic system, c-kit protein is expressed by 70% of CD34+ cells in BM, including lineage-restricted hematopoietic progenitor cells [30]. In addition, CD34 expression is lost during differentiation in the hematopoietic lineage, and it is maintained in the angioblastic one [31]. Further molecular studies are recommended for more conclusive outcome.

From the current observation, we can conclude that HSCs are defective in CDLE cases as proved by CD34 and c-kit immunostaining. Further research is needed to investigate BM HSC in these cases and the underlying molecular mechanisms for such defect. Experimental studies using HSC transplantation for resistant or disseminated CDLE cases may be evaluated. And now questions arise, can we use HSC transplantation as a therapeutic option for resistant and disseminated CDLE cases after being used to treat SLE for decades? Is HSC transplantation cost-effective? What is the mechanism(s) of post-transplantation remission? Which, if any, patients may be cured by an autologous graft and which will require an allograft?


  Conclusion Top


We can conclude that HSCs are defective in CDLE cases as proved by CD34 and c-kit immunostaining. Further research is needed to investigate bone marrow HSCs and the underlying molecular mechanisms for such defect. Experimental studies using HSC transplantation for resistant or disseminated CDLE cases may be evaluated.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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