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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 36  |  Issue : 2  |  Page : 39-42

Expression of hair follicle stem cell markers CD34 and CK15 in scarring alopecia


1 Department of Pathology, St John’s Medical College & Hospital, Bangalore, Karnataka, India
2 Department of Dermatology, St John’s Medical College & Hospital, Bangalore, Karnataka, India

Date of Submission10-Mar-2016
Date of Acceptance31-Oct-2016
Date of Web Publication21-Mar-2017

Correspondence Address:
Rajalakshmi Tirumalae
Department of Pathology, St John’s Medical College & Hospital, Bangalore 560 034, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-6530.202643

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  Abstract 


Introduction Pathogenesis of scarring alopecia (SA) is poorly understood with recent studies implicating loss of follicle stem cells (FSC).
Objectives The aim of the present study was to examine the expression patterns of FSC markers CK15 and CD34 in SA and to compare them with biopsies from normal and non-SA cases.
Materials and methods Twenty-one cases of SA were assessed using CK15 and CD34 immunostains. Biopsies from normal scalp and alopecia areata (AA) cases were studied for comparison.
Results CD34 was expressed in the outer root sheath of the lower segment of follicles in all normal, 33% of the AA and 33% of the SA cases. CK15 was seen in the outer root sheath of the upper segment of hair follicles in 100% of normal and AA biopsies, compared with 91% of SA. CK15 was expressed in the lower segment of the follicle in 9/10 cases of SA, which is hitherto undescribed. CK15 was also retained in the bulge in 84% of the SA cases.
Conclusion CD34 FSCs are lost in most SA cases. CK15 FSC are retained in both the bulge and the rest of the follicle, signifying that stem cell destruction alone does not eventuate in hair loss. The aberrant distribution of CK15 also suggests that there may be compensatory mechanisms yet to be understood.

Keywords: CD34, CK15, follicle stem cells, scarring alopecia


How to cite this article:
Yeliur IK, Tirumalae R, Bhat I, Abraham A. Expression of hair follicle stem cell markers CD34 and CK15 in scarring alopecia. Egypt J Dermatol Venerol 2016;36:39-42

How to cite this URL:
Yeliur IK, Tirumalae R, Bhat I, Abraham A. Expression of hair follicle stem cell markers CD34 and CK15 in scarring alopecia. Egypt J Dermatol Venerol [serial online] 2016 [cited 2020 May 28];36:39-42. Available from: http://www.ejdv.eg.net/text.asp?2016/36/2/39/202643




  Introduction Top


Alopecias are a challenge to both pathologists and dermatologists. They are separated into scarring/cicatricial and non-scarring forms according to the NAHRS classification [1]. Scarring alopecia (SA) is an irreversible process, and sometimes, even non-scarring forms show poor response to treatment. There has been a lot of interest in the role played by hair follicle stem cells (FSC) in the pathogenesis and regeneration in SA [2],[3]. These have been studied by the expression of FSC markers such as cytokeratin 15 (CK 15), CD34 and nestin in various parts of the hair follicle [4],[5],[6]. The published data is dominated by the west and there are no studies exploring this avenue among Indian patients. The objectives of the study were (a) to examine the expression patterns of hair stem cell markers CK 15 and CD34 in SA and (b) to compare the above with the biopsies of normal scalp and those with non-SA.


  Materials and methods Top


This cross-sectional study included 21 cases signed out as SA (12 lichen planopilaris, 5 lupus erythematosus and 4 cases of late-stage SA unclassified) by the pathologist where clinical data, slides and sufficient tissue were available. For comparison, we selected five cases of normal scalp (biopsy was carried out for an unrelated condition such as nevus, seborrhoeic keratosis) and six cases of alopecia areata (AA). Institutional ethical clearance was obtained.

Representative haematoxylin and eosin-stained sections were recut from the tissue blocks to ensure that follicles were present. Sections of 4-µm thickness were cut from the paraffin blocks. Immunohistochemistry was carried out on these using primary antibodies to CD34 (clone QBEND/10; Biogenex, California, USA) and CK 15 (clone EPR-1614Y; Biogenex), and visualized using the labelled streptavidin-biotin method.

Expression of CD34 and CK15 were assessed in different parts of the hair follicle, that is, outer root sheath (ORS) at isthmus and infundibulum, that is, upper segment (US), ORS below isthmus, that is, lower segment (LS), and bulge. CD34 and CK15 were considered positive when at least 10% of the outermost cells of the ORS showed cytoplasmic/membranous staining. We used the 10% cutoff based on the study by Pozdnyakova and Mahalingam [7], where most cases showed staining in the range of 10 to >50%. Only anagen follicles were assessed, as catagen and telogen follicles do not have an LS.

Normally, CD34 is expressed in the LS of the follicle and CK15 in the US. CK15 is also expressed by the bulge cells and variably in the epidermis [8].

The slides were analysed by two pathologists, blinded to the histopathologic diagnosis. Results were tabulated. Fisher’s exact test was used to calculate the statistical significance at a P-value of less than 0.05.


  Results Top


The results of the immunohistochemical stains are tabulated in [Table 1].
Table 1 Expression of follicle stem cell markers CD34 and CK15 in normal scalp, alopecia areata and scarring alopecia

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Expression of CD34 in the LS of follicles was seen in all normal biopsies in contrast to 33% of AA and 33% of SA cases ([Figure 1]). Both AA and SA cases showed loss of expression of CD34 in comparison with normal cases ([Figure 2]), but statistical significance was seen in SA (P=0.01). CK15 was expressed in the US of follicles in all normal, AA and SA cases ([Figure 2]). Expression of CK15 was also noted in the LS of the follicles in nine out of 10 SA cases, whereas none of the normal follicles or AA follicles showed LS staining ([Figure 2]). This expression was statistically significant (P=0.002). The lower portion of the follicle could not be visualized in the remaining 11 SA cases.
Figure 1 Normal scalp: (a) shows CK15 positive cells (arrow) in the outer root sheath (ORS) of the upper segment (US) of the follicle (CK15, ×100); (b) shows CD34 positive cells (arrow) in the ORS of the lower segment (LS) of the follicle (CD34, ×100).

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Figure 2 Scarring alopecia: (a) shows CK15 positive cells in the outer root sheath (ORS) of the upper segment (US) of the follicle as well as in the lower segment (LS) (CK15, ×100); (b) shows the absence of CD34 positive cells in the follicle (CD34, ×100).

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The cells of the bulge expressed CK15 in all normal and AA cases ([Figure 3]). In SA, the bulge was visualized in 13 cases, of which 11 showed positive staining with CK15.
Figure 3 Section showing the bulge with CK15 positive cells in a normal scalp (CK15, ×200).

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  Discussion Top


By the 16th week of gestation, the developing hair follicle germ shows three distinct hemispherical protrusions. The uppermost gives rise to the apocrine unit, the middle one forms the sebaceous unit and the lowermost bulge (or wulst) serves as the site of attachment of the arrector pili muscle [9]. The ‘bulge activation hypothesis’, which became popular in the 20th century, proposed that the bulge was home to multipotent stem cells [10]. Destruction of these cells in inflammatory processes was believed to lead to irreversible hair loss.

Liu et al. [4] discovered that the stem cells of the bulge are marked immunohistochemically by antibodies to CK15. CK15 is also expressed normally in the US of the follicle. Subsequent studies showed a loss of CK15 positive keratinocytes in various SA cases [11]. CD34, generally used as a vascular/mesenchymal marker, is expressed normally in the LS of the hair follicle [5]. Nestin is another marker that has been used and stains the infundibulum and the inner root sheath in the LS [6]. We decided to use CD34 and CK15 as their differential expression highlights FSC throughout the length of the follicle.

The expression patterns of CD34 and CK15 of the normal scalp in the present study are in agreement with earlier findings [5],[8]. Inoue and Yoshimura [12] have shown that there is a differential expression of various markers in FSC and they categorized them into nine different types. They also showed that CK15+ CD200+ CD34-FSC had a greater colony-forming ability in vitro as compared with CK15, CD200+ and CD34+ [12]. This suggests that the CK15+ cells are akin to the undifferentiated FSC that are multipotent, whereas CD34+ cells are more restricted in their capacity, similar to the ‘transit amplifying’ cells derived from the CK15+ population [2].

Hoang et al. [8] did not find the loss of CD34 and CK15 expression in non-SA. In contrast, CD34 expression was lost in 66% (assessed in the anagen follicles) of our AA cases. This needs to be verified in larger cohorts to understand its implications. There was no change in the CK15 expression.

A comparison of the expression patterns of CK15 and CD34 in the present study with previous literature is presented in [Table 2].
Table 2 Comparison of CK15 and CD34 expression in scarring alopecia between various studies

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Loss of CD34 expression in SA was noted in the present study (66%) and also in a study by Hoang et al. [8] (31%), implying that there is some degree of stem cell loss. Both studies affirm the preservation of CK15 expression in the US. A surprising finding that emerged in the current study was the expression of CK15 in the ORS of the LS of the follicle. This was not described by Hoang et al. [8], but when we studied the photographs published in their paper, we noted that CK15 expression was present in the LS of the follicles in SA. As stated earlier, the CD34+ population is more restricted compared with the CK15+ FSC. There may be two possibilities here: (a) the loss of CD34 may have caused the migration of CK15+ FSC from other niches; or (b) the local microenvironment may have triggered the FSC to switch to the CK15+ phenotype. Whatever the mechanism, it is clear that the loss of one phenotype of FSC is compensated by the other.

In a study by Pozdnyakova and Mahalingam [7], it was noted that there was a loss of CK15 in the bulge in SA cases associated with active disease and severe inflammation, whereas CK15 was normally expressed in the later, fibrotic lesions. The findings in our study ratify the above, as most of the cases in our series were late lesions. This implies that the loss of FSC of the bulge is not as crucial in SA as deemed earlier. In fact, the loss of FSC expression may be an effect rather than the cause and there appear to be mechanisms to restore these cells. A discussion of what other factors are at play is beyond the scope of this study.

Limitations of this study include the small number of cases studied. We did not study the association of stem cells with the extent of inflammatory cell infiltrate. Most of the cases we encountered were late lesions with minimal inflammation. In addition, the bulge could not be visualized in all the cases.


  Conclusion Top


There is some degree of loss of CD34+ FSC in SA in comparison with normal scalp and non-SA. However, there was no difference in the expression of CK15+ FSC among the three groups, even in the bulge region. This suggests that the loss of FSC alone may not explain the irreversible hair loss in patients with SA and other, as yet unexplored factors may be at play.

Financial support and sponsorship

This study was funded by Research Society, St. John’s Medical College, Bangalore, India.Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Olsen EA, Bergfeld WF, Cotsarelis G, Price VH, Shapiro J, Sinclair R et al. Summary of North American Hair Research Society (NAHRS)-sponsored Workshop on Cicatricial Alopecia, Duke University Medical Center, February 10 and 11, 2001. J Am Acad Dermatol 2003; 48:103–110.  Back to cited text no. 1
    
2.
Lavker RM, Sun TT. Epidermal stem cells. J Invest Dermatol 1983; 81:S121–S127.  Back to cited text no. 2
    
3.
Cotsarelis G. Epithelial stem cells: a folliculocentric view. J Invest Dermatol 2006; 126:1459–1468.  Back to cited text no. 3
    
4.
Liu Y, Lyle S, Yang Z, Cotsarelis G. Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge. J Invest Dermatol 2003; 121:963–968.  Back to cited text no. 4
    
5.
Poblet E, Jimenez F, Godinez JM et al. The immunohistochemical expression of CD34 in human hair follicles: a comparative study with the bulge marker CK15. Clin Exp Dermatol 2006; 31:807–812.  Back to cited text no. 5
    
6.
Li L, Mignone J, Yang M et al. Nestin expression in hair follicle sheath progenitor cells. Proc Natl Acad Sci USA 2003; 100:9958–9961.  Back to cited text no. 6
    
7.
Pozdnyakova O, Mahalingam M. Involvement of the bulge region in primary scarring alopecia. J Cutan Pathol 2008; 35:922–925.  Back to cited text no. 7
    
8.
Hoang MP, Keady M, Mahalingam M. Stem cell markers (cytokeratin 15, CD34 and nestin) in primary scarring and nonscarring alopecia. Br J Dermatol 2009; 160:609–615.  Back to cited text no. 8
    
9.
Ackerman AB, Böer A, Bennin B, Gottlieb GJ. Embryologic, histologic and anatomic aspects in histologic diagnosis of inflammatory diseases of the skin. 3rd ed. New York City: Ardor Scribendi Ltd.; 2005.  Back to cited text no. 9
    
10.
Sun TT, Cotsarelis G, Lavker RM. Hair follicular stem cells: the bulge-activation hypothesis. J Invest Dermatol 1991; 96:77S–78S.  Back to cited text no. 10
    
11.
Al-Refu K, Edward S, Ingham E, Goodfield M. Expression of hair follicle stem cells detected by cytokeratin 15 stain: implications for pathogenesis of the scarring process in cutaneous lupus erythematosus. Br J Dermatol 2009; 160:1188–1196.  Back to cited text no. 11
    
12.
Inoue K, Yoshimura K. Isolation and characterization of human hair follicle epithelial cells. Methods Mol Biol 2013; 946:411–421.  Back to cited text no. 12
    


    Figures

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

  [Table 1], [Table 2]



 

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