serum Concentrations of interferon-gamma (iFn-g) in patients with ...

Serum Concentrations of Interferon-gamma (IFN-g) in Patients with Alopecia areata: Correlation with clinical type and duration of the disease

ORIGINAL PAPER

Serum Concentrations of Interferon-gamma (IFN-g) in Patients with Alopecia areata: Correlation with clinical type and duration of the disease Emina Kasumagic-Halilovic1, Asja Prohic1, Jasenko Karamehic2 Department of Dermatovenerology, Sarajevo University Clinical Center Sarajevo, Bosnia and Herzegovina1 Institute of Clinical Immunology, Clinical Centre of University of Sarajevo, Bosnia and Herzegovina2

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lopecia areata (AA) is a heterogeneous disease characterized by nonscarring hair loss on the scalp or other parts of the body. A wide range of clinical presentations can occur-from a single patch of hair loss (alopecia unilocularis, AUl), multiple patches (alopecia multilocularis, AM) to complete loss of hair on the scalp (alopecia totalis, AT) or the entire body (alopecia universalis, AU). The cause of AA is unknown although most evidence supports the hypothesis that AA is a T-cell mediated autoimmune disease of the hair follicle and that cytokines play an important role. The aim of the study was to evaluate serum concentrations of interferon-gamma (IFN-g) in patients with AA and the healthy subjects and also to assess a possible association between IFN-g and clinical type and duration of the disease. Sixty patients with AA and 20 healthy controls were enrolled in the study. Serum concentrations of IFN-g were determined by ELISA method. The serum concentration of IFN-g in patients with AA was significantly higher than that in the control group (10.62±1.09 pg/mL vs 10.02± 0.62 pg/mL, respectively). Significantly elevated serum IFN-g were noticed in patients with AU type (11.81±1.11 pg/mL), expecialy those suffering from AT (12.30±0.93 pg/mL), compared with both patients with AUl (10.20±0.59 pg/mL) and patients with AM clinical type (10.21±0.78 pg/mL). There was no significant difference in serum IFN-g concentration between patients with AUl and AM group, as well as between patients with AT and AU. No correlations were found between duration of disease and the serum levels of IFN-g. Our findings confirm previously published data that the Th1 type cytokine IFN-g is elevated in the serum of AA patients. Key words: alopecia areata, interferon-gamma

shown that within the cascade of pathogenesis of AA, cytokines play a crucial role. The immune response presented in AA is associated with aberrant lesional expression of interferon-gamma (IFN-g), interleukin-2 (IL-2) and IL-1b, and overexpression of ICAM-1 i MHC molecules on hair follicle keratinocytes and dermal papilla cells (1). IFN-g is produced by perifollicular or follicular antigen presenting cells and among several actions it also deprives dermal papilla cells of their ability to maintain anagen hair growth (2). The changes in serum IFN-g concentrations were found in many diseases, such as psoriasis (3,4) and systemic lupus erythematosus (5). In some of these diseases, serum IFN-g concentration corelated with activity and intensity of the disease, and may be used as a prognostic factor. The aim of our study was to evaluate serum concentrations of IFN-g in patients with AA and healthy subjects and also to assess a possible association between IFN-g and clinical type and duration of the disease.

Corresponding author: Emina Kasumagic-Halilovic, MD, PhD, Department of Dermatovenerology, Clinical Centre of University of Sarajevo, 71 000 Sarajevo, Bosnia and Herzegovina, E-mail: kasumagicemina@yahoo. com

2. MATERIAL AND METHODS The study included 60 patients with AA (36 females and 24 males, median age 35.6). The patients who had received any treatment within previous 3 months were excluded from the study, as well as patients with any diseases based on the immune pathomechanism, which could influence serum concentrations of IFN-g. According to the clinical type of AA, patients were divided into 4 groups: AUl (n=13, 21.6%) patients with alope-

1. INTRODUCTION Alopecia areata (AA) is heterogeneous disease characterized by nonsccaring hair loss on the scalp or other parts of the body. A wide range of clinical presentation can occur-from a single patch of hair loss (alopecia unilocularis, AUl), multiple patches (alopecia multilocularis, AM) to complete loss of 212

hair on the scalp (alopecia totalis, AT) or the entire body (alopecia universalis, AU). The histopatologic features of the AA consist of perifollicular lymhocytic infiltrates around anagen hair follicles, consisting of both CD4+ and intrafollicular infiltrates of CD8+ cells. Although the etiopathogenesis of the disease is not clear, several studies have

MED ARH 2010; 64(4) • ORIGINAL PAPER


cia unilocularis, AM (n=33, 55%) patients with alopecia multilocularis, AT (n=7, 11.7%) total alopecia and AU (n=7, 11.7%) alopecia universalis. The control group consist of 20 generaly healthy people (11 female and 9 male, median age 32.6). Serum concentrations of IFN-g were measured by an enzyme-linked immunosorbent assay (ELISA) technique, using Quantikine Human IFN-g Immunoasay (R&D System, Minneapolis, MN, USA). Briefly, this assay empoys the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for IFN-g has been precoated onto a microplate. Standards and samples are pipetted into the wells and any IFN-g present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for IFN-g is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IFN-g bound in the initial step. The color development is stopped and the intensity of the color is measured. The data a re expressed as mean±standard deviation. The test distribution was done by KolmogorovSmirnov test, and comparations were performed by T-test. The data were considered statistically significant if p values were less than 0.05.

3. RESULTS The study group composed of 60 patients with AA (36 female and 24 male; the mean age of the patients was 35.6 years, ranging from 5 to 69 years), and 20 healthy controls (11 female and 9 male; the mean age 32.6 years, ranging from 6 to 63 years). There were no significant difference in age and female/ male ratio between the patients and controls (p>0.05). The mean duration of AA was 14.5±25.4 (range 1-119 months). In the total of 60 patients with AA, there were 13 (21.6%) patients with unilocular lesion, 33 (55%) with multilocular lesions, 7 (11.7%) with alopecia totalis and 7 (11.7%) with alopecia universalis. The serum concentration of IFN-g in patients with AA was significantly higher than that in the control group (10.62±1.09 pg/mL vs 10.02±0.62 pg/ mL, respectively). Significantly ele-

vated serum IFN-g concentrations were noticed in patients with AU type (11.81±1.11 pg/mL), expecialy those suffering from AT (12.30±0.93 pg/mL), compared with both patients with AUl (10.20±0.59 pg/mL) and patients with AM clinical type (10.21±0.78 pg/mL). There was no significant difference in serum IFN-g concentration between patients with AUl and AM group, as well as between patients with AT and AU. Patients with longer duration of the disease had higher concentration of IFN-g, but not significantly.

4. DISCUSSION Although the etiopathogenesis of AA is poorly understood, most researches think that it is connected with immune processes. It has been reported that various autoimmune diseases often coexist with AA, and various autoantibodies against different tissues, including those directly against hair follicle, have been noticed in AA patients (6-8). The specific targets of follicular autoantibodies seen in AA include multiple components of anagen hair follicles. The strongest evidence implicating autoimmune mechanism in the pathogenesis of AA has been provided by studies involving mice with severe combined immunodeficiency (SCID). In a set of experiments by Gilhar et al. , (911) AA was induced on human scalp explants transplanted onto SCID mice by injection of autologous T lymphocytes from lesional skin. In particular, only T lymphocytes cultured with hair follicle homogenate and antigen presenting cells were capable of inducing AA in scalp explants. Both the clinical and histopathological features of AA were reproduced in this model, including hair loss, perifollicular T lymphocyte infiltration, and expression of HLADR and ICAM-1 in the follicular epithelium. This suggest that AA is mediated by T-cells, which recognize a hair follicle antigen . Cytokines play an important role in both physiology and patophysiology of human skin, and it is possible that thay coordinate the cyclical hair growth. Hair loss may occur because proinflammatory cytokines interfere with the hair cycle, leading to premature arrest of hair cycling with cessation of hair growth (12). IFN-g is the main cytokine known to be aberrantly expressed in AA

though a CD4+ Th1 mediated response. By using immunohistochemical and in situ hybridization studies to demonstrate the persistence of proinflammatory as well as apoptotic mechanisms in the skin biopsies from patients with chronic AA, Bodemer et al. have confirmed the presence of a cellular infiltrate in close contact with the hair follicle, producing IFN-g in association with proinflammatory cytokine production (13). IFN-g is produced by perifollicular or follicular antigen presenting cells and among several actions it also deprives dermal papilla cells of their ability to maintain anagen hair growth (2). Ito et al. reported that IFN-g rapidly inhibited hair elongation in cultured human anagen hair follicles and induced morphological signs of catagen transformation after only four days of culture, faster than with other reported catagen-inducers (14). Proliferation was inhibited, apoptosis was increased, and follicular melanogenesis was switched of in hair bulb keratinocytes treated in situ with IFN-g. To determine which cytokines may be involved with overexpression of ICAM-1 and MHC molecules on dermal papilla cells of affected hair follicles, Konig et al. were able to imitate the in vivo situation of AA (1). They found that incubation with IFN-g led to a time-dependent upregulation of the surface molecules, as well as to an overexpression of ICAM-1. In addition, increased serum levels of IFN-g in patients with AA compared with normal controls has been reported, further suggesting a role for this cytokine. The results presented in our study demonstrate that the mean serum levels of IFN-g were significantly elevated in AA patients in comparison to healthy subjects. And also the serum levels of IFN-g in patients with AT and AU were significantly increased. These results are consistent with a clinical study performed by Arca et al. (15). They compared the serum levels of IFNg in patients with AA and the control group and also they investigatived the difference between the localized form of the disease with the extensive forms like AT and AU. It has been shown that serum levels of IFN-g are significantly higher in patients with AT or AU compared to controls, but no significant difference has been found in levels of IFNg between localozed AA and those with


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more extensive forms. Our findings are similar to the study of Lortkipanidze et al. , who also recorded a significant increase in serum IFN-g in patients with AA (16). In the study of Teraki et al. (17), they compared the serum levels of cytokines, including IFN-g, TNF-a, IL-1a, IL-2, IL-4 and IL-6 in patients with the localized form and the extensive form and found that the serum levels of IL-1a and IL-4 were significantly elevated in patients with the lokalized form. In contrast, the serum levels of IFN-g and IL-2 were significantly elevated in patients with the extensive form. They said that these findings could be interpreted as an indication that Th1 type cytokines might be critical for the progression to the extensive form and that Th2 type cytokines may exert a more subtle influence on the inhibition of a cell-mediated attak on hair follicles. After that, Barahmani et al. analyzed serum cytokine profiles in 269 patients with AA and found it that increased IFN-g levels is associated with AA regardless of disease severity (18).

5. CONCLUSION In conclusion, IFN-g seems to be a useful indicator of the activity of AA and that it may play an important role in the development of this disease. Further investigations are required to clarify the pathogenic role and clinical significance of IFN-g, and these findings may provide important clues to assist in the development of new therapeutic strategies for patients with AA.

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