|Year : 2022 | Volume
| Issue : 1 | Page : 54-59
T-helper and natural killer cells’ disturbance in active alopecia areata
Abdel-Khalek H Younes1, Refaat R Hammad1, Ali Sobhy2
1 Department of Dermatology, Andrology & STIs, Faculty of Medicine, Al-Azhar University, Assiut, Egypt
2 Clinical Pathology, Faculty of Medicine, Al-Azhar University, Assiut, Egypt
|Date of Submission||04-Jul-2021|
|Date of Decision||28-Jul-2021|
|Date of Acceptance||16-Aug-2021|
|Date of Web Publication||4-Mar-2022|
Source of Support: None, Conflict of Interest: None
Background and aim Alopecia areata (AA) is a common nonscarring hair disease targeting the hair follicles during the anagen phase, leading to hair loss. The etiology of AA is still unclear; however, evidence revealed the association of this disease with immune disturbance. Therefore, we aimed in this study to evaluate the level of natural killer (NK) and T-helper 1 (Th1) cells in the peripheral circulation of patients with different subtypes of AA during the active stage in comparison with the controls.
Patients and methods Venous blood samples from 50 female patients and 50 healthy-controls were collected to evaluate the level of Th1 and NK cells using flow cytometry.
Results Patients with active patchy AA, alopecia totalis, and alopecia universalis were associated with a lower level of circulating Th1 and NK cells when compared with the controls.
Conclusion The observed reduction in the circulating Th1 and NK cells indicates the autoimmune nature of the disease. Also, we highlight the cellular shift from the peripheral circulation to the lesion site during the active stage; however, more research is required to reveal the exact mechanism for this phenomenon.
Keywords: alopecia areata, natural killer cells, T-helper cells
|How to cite this article:|
Younes AKH, Hammad RR, Sobhy A. T-helper and natural killer cells’ disturbance in active alopecia areata. Al-Azhar Assiut Med J 2022;20:54-9
|How to cite this URL:|
Younes AKH, Hammad RR, Sobhy A. T-helper and natural killer cells’ disturbance in active alopecia areata. Al-Azhar Assiut Med J [serial online] 2022 [cited 2022 Jun 29];20:54-9. Available from: http://www.azmj.eg.net/text.asp?2022/20/1/54/339079
| Introduction|| |
Alopecia areata (AA) is a widespread disease marked by the presence of noncicatricial hair loss of the scalp and other parts of the body . AA targets about 2% of the population with equal incidence in both sex and for all age groups .
AA disease is marked by a wide variety of clinical subtypes, it may affect only the scalp hair follicle in a patchy manner, also, it can progress to involve the whole follicle of the scalp or even it can affect all follicles all over the body .
The etiopathogenesis of AA is still a weird process, however, evidence points to different types of immune cells and cytokines playing a critical role in the occurrence of the disease .
Lesional biopsy revealed the abnormal disturbance and a high immune-cell density around and inside the hair follicle, which are more clearly obvious during the active stage of this disease . Recent studies strongly implicate CD4+, CD8+, and natural killer (NK) in the etiopathogenesis of AA . The peribulbar clustering and follicular infiltration of Th, CD8+, and NK cells, leads to the collapse of the immune privilege .
Due to the systemic autoimmune nature of this disease, it was noticed that the lesional site disturbance of inflammatory cells accompanied also by a disturbance of peripheral circulation immune cells. So, we aimed in this study to assess the level of disturbance of the immune cell during the active stage in different subtypes of AA disease.
| Patients and methods|| |
Venous blood samples were collected from 50 females diagnosed with AA and 50 healthy-controls with age ranging from 20 to 45 years at the Outpatient Clinic of Dermatology, Venereology and Andrology, Al-Azhar University, Assiut Governorate, during the period between October 2019 and December 2020. The study was approved by the Committee of Local Institutional Ethics of Faculty of Medicine, Al-Azhar University, and informed written consent was obtained from all participants. The study is conducted in accordance with Helsinki standards as revised in 2013.
Patients with a confirmed diagnosis of active AA, were included, while patients with other autoimmune disorders or who were using immunosuppressive drugs were not allowed to participate in this study.
The activity of the disease was detected clinically by the pull test and investigated using the dermoscope.
Venous blood samples were collected from the participants and analyzed using Navios flow cytometer (Beckman Coulter, South Kraemer Boulevard, Brea, CA, USA). CD4, CD16, and CD56 expression were evaluated and presented as a percentage of total lymphocytes.
Patients were divided clinically according to their AA subtypes into patchy alopecia areata (PAA), alopecia totalis (AT), and alopecia universalis (AU).
The sample size was calculated using Stata/IC, version 16.1 for Windows (StataCrop LLC, College Station, Texas, USA). After reviewing the literature, we found that sample sizes of 100 patients fulfill at least 80% power to detect a difference of 5.9 between the null hypothesis that the mean of T-helper 1 (Th1) cells for both patients and control group is 40.5 and our hypothesis that the mean of Th1 cells is 34.6 with computed standard deviations of 10.4 for both groups. A two-sided two-sample t test with a level of significance equal to 0.05 was used.
Statistical analysis was done using Stata/IC, version 16.1. The test of normality was calculated by the Kolmogorov–Smirnov test. We used Kruskall–Wallis test to compare the mean between the non-normally distributed variables, while in the case of normally distributed variables, analysis of variance test was used. P value less than 0.05 was considered significant.
| Results|| |
Twenty-four female patients with PAA, 15 patients with AT, 11 patients with AU, and 50 healthy female controls were evaluated in this study. The demographic data of our studied population are shown in [Table 1].
|Table 1 Comparison between demographic data of alopecia areata subtypes and control|
Click here to view
The number of Th1 cells in the peripheral circulation of patients and controls was investigated. We found that the proportion of circulating Th1 cells was significantly lower in the peripheral circulation of different AA subtypes (P=0.006) when compared with the controls as shown in [Table 2] and [Figure 1].
|Table 2 Immunophenotype of the T-helper 1 and natural killer cells between different alopecia areata subtypes patients and control|
Click here to view
|Figure 1 Immunophenotype of the T-helper-1 cells among patients with different AA subtype and controls. AA, alopecia areata.|
Click here to view
Furthermore, the proportion of NK cells_CD56(bright) CD16(dim) in the peripheral circulation of patients manifested a lower significant level when compared with control P value of 0.018 as shown in [Table 2] and [Figure 2].
|Figure 2 Immunophenotype of the NK cells_CD56(bright) CD16(dim) (%) among patients with different AA subtypes and controls. AA, alopecia areata; NK, natural killer.|
Click here to view
On the other hand, there was no significant difference regarding the proportion of neither NK cells_CD56(bright) CD16(+) (%) nor NK cells_CD56(dim) CD16(+) cells as shown in [Table 2].
As regards the comparison of the immunophenotype of Th1 and NK cells among the AA-subtype patients, there were no significant differences between the groups as shown in [Table 3].
|Table 3 Comparison between the immunophenotype of the T helper and natural killer cells in alopecia areata subtypes patients|
Click here to view
To detect the presence of any potentially confounding variables between patients and controls, we conducted univariate and multivariate logistic regression analysis.
We found that the AA patients associated with significant lower Th1 cells with odds ratio (OR) 0.93 in comparison with control and still with a significant lower OR (0.94) after uncovering the influence of other confounders by using the adjusted model. Also, NK cells_CD56(bright) CD16(dim) (%) showed a significant lower OR in the unadjusted and adjusted model with OR (0.41) and (0.44), respectively, as shown in [Table 4].
|Table 4 Univariate and multivariate logistic regression analyses showing the associations between laboratory parameters and alopecia areata|
Click here to view
| Discussion|| |
AA is a common nonscaring hair disorder targeting the hair follicles. The pathogenesis of AA is still unclear, despite the accumulated evidence about the autoimmune nature of this disease.
Previous studies about AA reported a wide age and sex-incidence variety. Fricke and Miteva  reported that AA disease can occur at any age and any sex, however, Ejaz et al.  reported the peak incidence in the second decade of life. Our study result is matched with Al-Ajlan et al.  as they reported that the incidence is at its highest in the third decade of life and the disease is more common in females.
Our study was restricted only to the female sex to avoid the influence of the sex as a confounder on the outcome.
In this study, we noticed that AU associated with the longer disease duration followed by AT, and this comes in accordance with the studies done by Goh et al. , Tan et al. , and Qi et al.  as they reported that patients with AT and AU had a much longer disease duration than those with PAA.
In our study, we found that the recurrence rate was more common in AT and AU when compared with PAA, our results come in accordance with data reported by Qi et al. , as they returned that to the early age of onset and longer disease duration, which may be associated with a higher rate of recurrence.
T-helper-1 and T-cytotoxic cells represent the majority of T-lymphocytes. T-helper-1 cells are activated and differentiated to play a critical role in organizing the immune response .
Many previously conducted studies reported that the main etiopathogenesis of the AA disease is the disturbance of the immune cells either inside or around hair follicles. However, other studies found that the disturbance not only occurs at the site of the lesion but also it can affect the draining lymph node, spleen, and the peripheral circulation of the patients ,.
To the best of our knowledge, there are no recent studies showing a result regarding the disturbance of the Th1 and NK cells in the peripheral circulation of AA subtypes while the disease is in its active phase.
In our study, we found that during the active phase of this disorder, T-helper-1 and NK cells were found with a less level in the peripheral circulation of AA subtypes when compared with healthy participants.
In a previous study conducted by Lee et al.  without specifying the AA subtypes, they also reported a less level of Th1 in AA patients than the controls. In contrast, in the study done by Lutz et al. , they reported that there was no significant difference between the patients when compared with healthy participants.
Studies conducted by Zöller et al. , Kubo et al. , and Hamed et al. , helped us to explain the noticed reduction of the T-helper cells in the circulation of patients during the active stage of the disease. They found during the early active phase of this disease that there is an increase in the proportion of T-regulatory cells that prevent the proliferative activity of T-helper 1, however at the site of the lesion, they noticed the reduced level of T-regulatory cells explaining the increased number of Th1 around the hair follicles.
NK cells are classified, depending on their surface markers into CD16 and CD56. CD56(bright) CD16(dim/)(−) representing about 10% and CD56(dim) CD16(+) representing about 90% of the circulating NK cells are considered the major NK-cell subtypes .
CD56(bright) CD16- NK display a more important immunomodulatory role, and considered the most efficient cytokine producers’ cells, many cytokines are released by those cells, including, interleukin-10, interleukin-13, colony-stimulating factor 2, tumor necrosis factor-α, and interferon-γ .Paolini et al.  declared that NK-cells’ immunoregulatory abilities are mediated not only by cell-to-cell contact, but also by the soluble substances they release, which allow them to recruit and activate additional immune cells.
In this study, we investigated the nature and the frequency of NK cells in the peripheral blood of AA patients and their association with the activity of AA. Interestingly, we found a significant lower proportion of the NK cells_CD56(bright) CD16(dim) in the peripheral circulation of AA patients when compared with healthy participants. Our result is matched with the study done by Lutz et al. , however, being worked on the Fc immunoglobulin G receptor, not the CD56 and CD16 surface markers.
The reduction in the proportion of NK cells can be explained by the work of Lima et al. as they found that NK cells can travel via the blood and reach the hair follicles by using adhesion molecules and chemokine receptors. The expression of CXCR3/CCR5 on CD56bright NK cells forces them to move toward the affected tissue.
By multivariate-regression analysis, we found that lymphocyte count, T-helper, and NK cells are significantly disturbed in the peripheral blood of AA patients when compared with normal healthy participants.
To sum up our results, this work demonstrated that the disturbance of the lymphocytes and NK cells plays an important role in the pathogenesis of AA. Immunomodulating drugs could be used to block the migration of the immune cells from the bloodstream to the targeted hair follicles and can be effective in normalizing the disturbance of those immune cells during the early active stage of this disease. One of the limitations of this study is that the scalp biopsy was not done. Infiltrating T-helper and NK cells could be better studied using skin-tissue sections. The results can then be compared with peripheral blood samples to determine the cause of the observed changes in circulating peripheral T cells and NK cells.
Further studies comparing the histopathological change in the immune cells at the site of the lesion with the level of disturbance in the peripheral blood are needed, also, other studies are needed to clarify the clinical importance of our speculations.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Hosking AM, Juhasz M, Atanaskova Mesinkovska N. Complementary and alternative treatments for alopecia: a comprehensive review. Skin Appendage Disord 2019; 5:72–89.
Fricke ACV, Miteva M. Epidemiology and burden of alopecia areata: a systematic review. Clin Cosmet Investig Dermatol 2015; 8:397.
Pratt CH, King LE, Messenger AG, Christiano AM, Sundberg JP. Alopecia areata. Nat Rev Dis Primers 2017; 3:1–17.
Trüeb RM, Dias MFRG. Alopecia areata: a comprehensive review of pathogenesis and management. Clin Rev Allergy Immunol 2018; 54:68–87.
Dy LC, Whiting DA. Histopathology of alopecia areata, acute and chronic: why is it important to the clinician Dermatol Ther 2011; 24:369–374.
Guo H, Cheng Y, Shapiro J, McElwee K. The role of lymphocytes in the development and treatment of alopecia areata. Expert Rev Clin Immunol 2015; 11:1335–1351.
Bertolini M, McElwee K, Gilhar A, Bulfone-Paus S, Paus R. Hair follicle immune privilege and its collapse in alopecia areata. Exp Dermatol 2020; 29:703–725.
Ejaz A, Jameel K, Suhail M. Pattern and profile of alopecia areata in Pakistan. J Pak Assoc Dermatol 2016; 19:136–140.
Al-Ajlan A, Alqahtani ME, Alsuwaidan S, Alsalhi A. Prevalence of alopecia areata in Saudi Arabia: cross-sectional descriptive study. Cureus 2020; 12:9.
Goh C, Finkel M, Christos P, Sinha A. Profile of 513 patients with alopecia areata: associations of disease subtypes with atopy, autoimmune disease and positive family history. J Eur Acad Dermatol Venereol 2006; 20:1055–1060.
Tan E, Tay YK, Goh CL, Chin Giam Y. The pattern and profile of alopecia areata in Singapore − a study of 219 Asians. Int J Dermatol 2002; 41:748–753.
Qi S, Xu F, Yang Q, Dai W. Profile of alopecia areata in 655 Chinese patients: P2102. J Am Acad Dermatol 2010;62:3.
Luckheeram RV, Zhou R, Verma AD, Xia B. CD4+ T cells: differentiation and functions. Clin Dev Immunol 2012; 2012:925135.
Paus R, Bertolini M. The role of hair follicle immune privilege collapse in alopecia areata: status and perspectives. J Investig Dermatol Symp Proc 2013;16:S25–27.
Zöller M, McElwee KJ, Vitacolonna M, Hoffmann R. Apoptosis resistance in peripheral blood lymphocytes of alopecia areata patients. J Autoimmun 2004; 23:241–256.
Lee WS, Lee IW, Lee CH. A study on the changes of T lymphocyte of the peripheral blood in patients with alopecia areata using flow cytometry. Korean J Dermatol 1996; 34:600–607.
Lutz G, Niedecken H, Bauer R, Kreysel HW. Natural killer cell and cytotoxic/suppressor T cell deficiency in peripheral blood in subjects with alopecia areata. Austr J Dermatol 1988; 29:29–32.
Kubo R, Muramatsu S, Sagawa Y, Saito C, Kasuya S, Nishioka A et al.
Activated regulatory T cells are increased in patients with alopecia areata for suppressing disease acitivity. J Dermatol Sci 2017; 86:e27–e28.
Hamed FN, Åstrand A, Bertolini M, Rossi A, Maleki-Dizaji A, Messenger AG et al.
Alopecia areata patients show deficiency of FOXP3+CD39+ T regulatory cells and clonotypic restriction of Treg TCRβ-chain, which highlights the immunopathological aspect of the disease. PLoS ONE 2019; 14:e0210308-e.
Poli A, Michel T, Thérésine M, Andrès E, Hentges F, Zimmer J. CD56bright natural killer (NK) cells: an important NK cell subset. Immunology 2009; 126:458–465.
Paolini R, Bernardini G, Molfetta R, Santoni A. NK cells and interferons. Cytokine Growth Factor Rev 2015; 26:113–120.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]