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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 40  |  Issue : 2  |  Page : 84-91

Immunohistochemical study assessing stem cell factor receptor C-kit in patients with alopecia


1 Department of Dermatology and Venereology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission28-Jul-2019
Date of Acceptance19-Dec-2019
Date of Web Publication09-Jun-2020

Correspondence Address:
Nashwa Elfar
Department of Dermatology and Venereology, Faculty of Medicine, Tanta University, Tanta, 3111
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejdv.ejdv_40_19

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  Abstract 


Background Stem cell factor receptor c-kit is a transmembrane protein with tyrosine kinase activity encoded by the oncogene c-kit. The stem cell factor/c-kit signaling may be involved in the pathological processes of alopecia.
Objective The aim was to elucidate the role of the stem cell factor receptor c-kit in the pathogenesis of some types of alopecia.
Patients and methods The study included 80 patients (52 had alopecia areata and 28 had androgenetic alopecia), in addition to 40 normal healthy persons who served as a control group. Skin biopsies were obtained from each participant and stained with hematoxylin and eosin and stem cell factor receptor c-kit.
Results There was a statistically significant increase in intensity and count of stem cell factor receptor c-kit-positive cell expression in alopecia areata and androgenetic alopecia than in normal control specimens. There was a positive correlation between the intensity and count of stem cell factor receptor c-kit-positive cells in relation to the severity of alopecia.
Conclusions Stem cell factor receptor c-kit was implicated in the pathogenesis of alopecia areata and androgenic alopecia, and it could have a predictive role in the progression of alopecia in high-risk patients.

Keywords: alopecia areata, androgenic alopecia, stem cell factor receptor C- kit


How to cite this article:
Mourad B, Hasby E, Elfar N. Immunohistochemical study assessing stem cell factor receptor C-kit in patients with alopecia. Egypt J Dermatol Venerol 2020;40:84-91

How to cite this URL:
Mourad B, Hasby E, Elfar N. Immunohistochemical study assessing stem cell factor receptor C-kit in patients with alopecia. Egypt J Dermatol Venerol [serial online] 2020 [cited 2020 Sep 23];40:84-91. Available from: http://www.ejdv.eg.net/text.asp?2020/40/2/84/286285




  Introduction Top


Tissue stem cells are the body’s master cells with the ability to differentiate into tissue-specific precursor and specialized cells; the former cells actually proliferate, create tissue bulk, and play a critical role in maintenance of tissue structures and homeostasis [1]. The stromal cells produce and release the growth factor called stem cell factor, which stimulates cells expressing the stem cell factor receptor c-kit [2].

The c-kit receptor (CD117) is a transmembrane protein with tyrosine kinase activity encoded by the oncogene c-kit. It is an important member of type III receptor tyrosine kinase family. It is localized on the cell surface of hematopoietic stem cells and other cell types and acts as a receptor for stem cell factor. The binding of stem cell factor to c-kit transactivates the receptor tyrosine kinase to activate stem cells, and the stem cell factor/c-kit signaling induces cell survival, differentiation, and proliferation [2].

Stem cell factor receptor c-kit is important for gametogenesis, hematopoiesis, melanogenesis, and mast cell biology. Dysregulation of stem cell factor receptor c-Kit function is oncogenic, and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology [3].

In hair follicle, the cells of the keratinocytic and melanocytic lineage are responsible for hair growth and color, respectively, and express stem cell factor receptor c-kit [2]. Stem cell factor receptor c-kit may be responsible for hair growth as well as hair pigmentation [4].

The aim of this work was to elucidate the role of the stem cell factor receptor c-kit in the pathogenesis of some types of alopecia.


  Patients and methods Top


The current case–control study included 80 patients who had alopecia in addition to 40 normal control skin specimens from age-matched and sex-matched healthy individuals. Patients were recruited from Dermatology and Venereology Department, Tanta University Hospitals. Ethical approval was obtained from Ethical Committee before commencement of the study with approval number 31264 (10/2014).

All participants included in the study were either newly diagnosed patients or untreated patients. Patients with any other dermatological or systemic diseases and pregnant and lactating females were excluded from the study:
  1. All participants were subjected to complete careful history taking and thorough general and dermatological examinations. Detailed dermatological examination of scalp was performed to assess clinical types of alopecia and to classify the studied patients clinically as follows:
  2. Group I included 52 patients who had alopecia areata, graded according to SALT score [5].
  3. Group II included 28 patients who had androgenic alopecia, comprising 22 male patients, who were assessed according to Hamilton–Norwood scale [6], and six female patients, who were assessed according to Sinclair scale [7].
  4. Group III included 40 normal healthy persons who served as controls.


After taking written informed consent, routine laboratory investigations were done for all participants. Punch skin biopsies of 3 mm were taken from each patient with alopecia, and normal control skin specimens from matched sites were taken during plastic surgical operations, and then fixed in 10% formalin, and routinely processed paraffin-embedded tissue sections (3–4 μ) were prepared on charged glass slides for the following:
  1. Hematoxylin and eosin staining was performed to confirm the diagnosis of alopecia and demonstrate the histopathological features of lesions.
  2. Immunohistochemical staining: the method used for immunostaining was the standard avidin biotin peroxidase complex using C-kit antibody (C-19): sc-168 rabbit polyclonal IgG antibody available from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).


The slides were subjected to subsequent steps of deparaffinization and rehydration. Antigen retrieval was done by boiling in citrate buffer saline at pH 6.0 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 hematoxylin as a counterstain. All slides were examined by image analysis Leica q win at the same magnification. For each specimen, four separate fields of view were evaluated:
  1. C-kit was expressed as brown cytoplasmic staining. The intensity of brown staining of positivity was graded [8] as negative (if <5% positive cells), mild, moderate, and strong intensity [9].
  2. Sections stained with hematoxylin and eosin and immunostained for c-kit were scanned and imported into a personal computer. The length of hair follicles (mm) in each section was measured, and the mean length of the follicles was calculated.
  3. The number of hair follicle epithelial cells immunoreactive for stem cell factor receptor c-kit was counted, and the mean number in a follicle was calculated and expressed [10].
  4. Multiple images were taken for each immunohistochemically stained section by Leica DM500 microscope [Leica Microsystems (Schweiz) AGMax Schmidheiny Strasse 201CH-9435 Heerbrugg (Switzerland)] with built-in Leica ICC50 digital camera. Images were then entered into Image J image analyzing system version # 1.50d. The region of interest was determined by defining our selection on RGB stalk and then adjusting the threshold in comparison with the original image. Measurements were limited to threshold and set to area fraction (percentage). The mean area percentage of staining of multiple images from the same section was then calculated.


Statistical analysis of the data [11]

Data were analyzed using IBM SPSS software package version 20.0 (IBM Corp., Armonk, New York, USA). Qualitative data were described using number and percentage. Quantitative data were described using range, mean, SD, and median. Comparison between different groups regarding categorical variables was tested using χ2-test. Correction for χ2 was conducted using Fisher’s exact test or Monte Carlo correction. For normally distributed data, comparison between two independent populations was done using independent t-test. Significance of the obtained results was judged at the 5% level.


  Results Top


Clinical results

The demographic data of the studied patients are summarized in [Table 1].
Table 1 Demographic data of the studied patients of alopecia areata and androgenic alopecia

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In patients with alopecia areata (group I), the age ranged from 16 to 42 years, with a mean of 30.77±7.47 years and a median age of 33 years; 22 patients were less than 30 years and 30 patients were more than 30 years, and female to male ratio was 6 : 7. However, in patients with androgenetic alopecia (group II), the age ranged from 20–45 years, with a mean of 33.43±6.99 years and median age of 34 years; eight patients were less than 30 years and 20 patients were more than 30 years, and female to male ratio was 3 : 11. In the control group (group III), the age ranged from 18 to 45 years, with a mean of 32.95±8.78 years and median age of 31.5 years; 20 persons were less than 30 years and 20 persons were more than 30 years, and female to male ratio was 1 : 3. There was no statistically significant difference regarding the age and sex of patients among patients with alopecia areata, patients with androgenetic alopecia, and control group (P=0.458 and 0.179, respectively).

Patients with alopecia areata (group I) were classified according to SALT score as follows: 36 (69.2%) patients were S1, 10 (19.3%) patients were S2, two (3.8%) patients were S4b, and four (7.7%) patients were S5. In androgenic alopecia group (group II), female patients were classified according to Sinclair scoring, which showed that two (33.3%) patients were grade III, two (33.3%) patients were grade IV, and two (33.3%) patients were grade V. However, Modified Hamilton score for male patients showed that four (18.18%) patients were grade II, eight (36.36%) patients were grade III, six (27.27%) patients were grade IV, and four (18.18%) patients were grade V.

Histopathological results

Hematoxylin and eosin stain

There was no statistically significant difference regarding the shortening of length of hair follicle between patients with alopecia areata and patients with androgenetic alopecia (P1=0.388), although it was much shorter in patients with androgenetic alopecia than in alopecia areata. However, there was a statistically significant decrease in length of hair follicle in alopecia areata and androgenetic alopecia in comparison with normal control specimens (P2<0.001, P3<0.001, respectively) ([Table 2], [Figure 1]a–c).
Table 2 Comparison between the different studied groups according to length of hair follicle

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Figure 1 (a): Normal control specimen showed normal length of hair follicle (black arrow) (H&E, ×100); (b) alopecia areata specimen showed shortening of length of hair follicle (black arrow) (H&E, ×100); (c) androgenic alopecia specimen showed marked shortening of hair follicle (black arrow) with perifollicular lymphocytic infiltrate (H&E, ×100). H&E, hematoxylin and eosin.

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Immunohistochemical staining

There was no statistically significant difference in intensity of stem cell factor receptor c-kit expression between alopecia areata (group I) and androgenetic alopecia (group II) (P1=0.386), although it is more intense in group II than in group I. There was a highly statistically significant increase in intensity of expression in alopecia areata and androgenetic alopecia patient specimens than in normal control specimens (P2<0.001 and P3<0.001, respectively).

There was no statistically significant difference regarding count of stem cell factor receptor c-kit-positive cells per hair follicle between alopecia areata (group I) and androgenetic alopecia (group II) (P1=0.325), although it was more in group II specimens. There was a highly statistically significant increase in count of stem cell factor receptor c-kit-positive cells in alopecia areata and androgenetic alopecia than in normal control specimens (P2<0.001 and P3<0.001, respectively) ([Table 3]).
Table 3 Comparison between the different studied groups according to intensity of stem cell factor receptor c-kit expression and count of positive cells per hair follicle

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There was no statistically significant difference in percentage of stained area between alopecia areata (group I) and androgenetic alopecia (group II), although it was higher in androgenetic alopecia (P=0.110). There was a statistically significant increase in percentage of stained area per hair follicle in alopecia areata and androgenetic alopecia than in normal control specimens (P=0.001) ([Figure 2]a–c).
Figure 2 (a): Normal control specimen showed a hair follicle with few stem cell factor receptor c-kit-positive cells and mild intensity of expression (immunoperoxidase, ×400); (b) alopecia areata specimen showed hair follicle with many c-kit-positive cells and moderate intensity of expression (immunoperoxidase, ×400); (c) androgenic alopecia specimen showed a hair follicle with many stem cell factor receptor c-kit-positive cells with moderate intensity of expression (immunoperoxidase, ×400).

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In patients with alopecia areata, there was a positive correlation between the intensity and count of stem cell factor receptor c-kit positive cells per hair follicle in relation to the severity of alopecia (P=0.181 and 0.752, respectively), (r=−0.065 and 0.271, respectively). However, there was a negative correlation between the intensity and count of stem cell factor receptor c-kit-positive cells and the length of hair follicle (P=0.113 and 0.303 respectively; r=−0.210 and −0.319, respectively).

In patients with androgenetic alopecia, there was a positive correlation between the intensity and count of stem cell factor receptor c-kit-positive cells per hair follicle in relation to the severity of alopecia (P=0. 327 and 0. 535 respectively; r=0.283 and 0.182, respectively). However, there was a negative correlation between the intensity and count of stem cell factor receptor c-kit-positive cells and the length of hair follicle (P=0.265 and 0.333, respectively; r=−0.320 and −0.280, respectively).


  Discussion Top


The stem cell factor receptor c-kit is a transmembrane protein with tyrosine kinase activity encoded by the oncogene c-kit. The ligand for c-kit is stem cell factor, a hematopoietic cytokine that plays an important role in maintaining the survival of hematopoietic cells [12].

The stem cell factor receptor c-kit is expressed in many tissues including mast cells, gastrointestinal stromal tumors, melanocytes in the skin, glial tumors, and interstitial cells of Cajal in the digestive tract and is a precise marker in the bone marrow for hematopoietic progenitor cells [13]. The bulge region of the hair follicle represents the best characterized epidermal stem cells population described to date, but there is evidence of other stem cells populations in the interfollicular epidermis, the sebaceous gland, and the dermis [14].

Quantitative immunohistochemistry detected the stem cell factor receptor c-kit in four focus areas: the epidermis, infundibulum, hair bulb (where pigment is formed), and mid-follicle outer root sheath [15].

In this study, the length of the hair follicle was significantly shorter in alopecia groups than normal control specimens. It was much shorter in androgenetic alopecia specimens than in alopecia areata specimens. These results agreed with the results of Ashrafuzzaman et al. [16], who noticed that the hair follicles tended to show a marked shortening of longitudinal length in alopecia areata and androgenetic alopecia as compared with the normal controls. They reported altered function of male hormones is involved in androgenetic alopecia.

The representative male hormone testosterone is produced in the testes and hematogenously transported into the dermal hair papilla, where 5α-reductase catalyzes the conversion of this hormone to 5α-dihydrotestosterone, which exerts more powerful effects. These male hormones reduce cell division activity and suppress hair growth activity [17].

Moreover, the longitudinal length of hair follicle as well as hair growth rate depends on the activity of the highly proliferative hair matrix cells in the hair bulb. The hair matrix cells are supplied by the stem cells existing in the bulge near the insertion of arrector pili muscle. These stem cells are activated by stem cell factor/c-kit signaling [16].

In this study, there was a significant increase in intensity of stem cell factor receptor c-kit expression in androgenetic alopecia and alopecia areata than in normal controls. The results of Ashrafuzzaman et al. [16] were in agreement with our study; they found stem cell factor receptor c-kit expression was mild in normal controls and marked in patients with androgenetic alopecia. They explained their results by reflecting a protective mechanism against potential downregulation of stem cell factor in stromal cells in dermal hair papilla.

Moreover, Peters et al. [18] noticed that disruption of the stem cell factor receptor c-kit/stem cell factor signaling pathway interferes with the survival, migration, and differentiation of melanocytes during generation of the hair follicle pigmentary unit. They explain these findings that melanoblasts express stem cell factor receptor c-kit as a prerequisite for migration into the stem cell factor-supplying hair follicle epithelium.

However, Randall et al. [15] reported that no significant difference in intensity of stem cell factor receptor c-kit expression was found between androgenetic alopecia and normal controls. They found that stem cell factor receptor c-kit expression was unaltered in balding follicles although that less stem cell factor is secreted. They explained their results by implication of other factors in activating stem cells.

In the current study, the count of stem cell factor receptor c-kit-positive cells per hair follicle and the percentages of stained area were highest in androgenetic alopecia and least in normal controls. Moreover, Ashrafuzzaman et al. [16] reported that the mean number of stem cell factor receptor c-kit-immunoreactive cells per hair follicle was significantly increased in the alopecia areata and androgenetic alopecia as compared with the normal controls and also increased in androgenetic alopecia as compared with alopecia areata.

Vanderford et al. [19] noticed that different types of nonscarring alopecia are stem cell factor receptor c-kit dependant. Sharov et al. [20] reported increase in stem cell factor receptor c-kit-positive cells in patients with chemotherapy-induced hair loss. They explained that stem cell factor/c-kit signaling may contribute to proliferation and migration of c-kit-positive follicular melanocytes into the epidermis after chemotherapy.

Bertolini et al. [21] mentioned that the number of proliferating stem cell factor receptor c-kit+ mast cells was increased in alopecia areata lesional skin compared with control skin. This result explained the reports that anti-histamines are beneficial in at least some patients with alopecia areata.

On the contrary, Randall et al. [15] detected no differences in stem cell factor receptor c-kit distribution, intensity of expression or cell concentration between normal and androgenetic alopecia follicles, but cultured dermal papilla cells from androgenetic alopecia follicles secreted less stem cell factor.

Bulge stem cells (hair follicles stem cells) are the essential prerequisite for the cyclic regeneration of hair follicles, during which they switch from phases of growth (anagen) via regression (catagen) to relative quiescence phase (telogen) [22].The upregulation of stem cell factor receptor c-kit in the hair follicle cells may reflect a protective mechanism against the potential downregulation of stem cell factor in stromal cells in dermal hair papilla. So it is clear that stem cell factor/c-kit signaling plays an important role in guiding and sustaining the development and growth of the hair follicle [16].

The quantitative PCR of human hair follicles indicated that stem cell factor receptor c-kit expression as well as the expression of several melanogenic factors was significantly lower in unpigmented than in pigmented follicles. These data revealed a vital role of stem cell factor/stem cell factor receptor c-kit signaling in the maintenance of human hair follicle melanogenesis during the anagen cycle and its involvement in physiological ageing of the hair follicle pigmentary unit [20].

Stem cell factor/stem cell factor receptor c-kit signaling is critical for melanocyte proliferation, differentiation, and/or migration during the hair cycle, and hence is required for cyclic regeneration of the hair pigmentation unit postnatally [21].

It could be concluded that the stem cell factor receptor c-kit was implicated in the pathogenesis of alopecia areata and androgenic alopecia, and it could have a predictive role in progression of alopecia in high-risk patients. Moreover, the length of hair follicles was much shorter in alopecia areata and androgenetic alopecia specimens than in normal specimens. This may suggest that longitudinal length of hair follicle as well as hair growth rate depends on the activity of the hair matrix cells in the hair bulb which are supplied by the stem cells existing in the bulge. These stem cells are activated by stem cell factor/c-kit signaling.

The limitations in our study were the small sample size of patients with different clinical types of alopecia. Moreover, there was a lack of prior research studies on the same topic of the current study, which reduced data comparison.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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