Untreated Alopecia Areata Research Articles

The profile of cytokines (IL-2, IFN-γ, IL-4, IL-10, IL-17A, and IL-23) in active alopecia areata.

Alopecia areata (AA) is an autoimmune disease due to aberrant T-cell response against hair follicle self-antigens. Previous studies support the role of Th1 cytokines in pathogenesis of AA, but the role of Th2, Th17, and Treg cytokines remains to be fully elucidated. To assess the serum levels of cytokines secreted by Th1 (IL-2, IFN-γ), Th2 (IL-4), Th17 (IL-23, IL-17A), and Treg (IL-10) pathways in patients of active AA and to correlate their levels with the severity of the disease. Forty patients with untreated active AA of the scalp and forty age- and sex-matched healthy controls were included. Serum levels of cytokines IL-2, IFN-γ, IL-17A, IL-23, IL-4, and IL-10 were measured using enzyme-linked immunosorbent assay. Serum levels of cytokines IL-2, IFN-γ, IL-17A, and IL-10 were significantly raised while serum levels of IL-23 were nonsignificantly raised in AA patients as compared to controls. The levels of IL-4 were significantly lower in AA patients as compared to controls. (P<0.05). Also, significant positive correlation was found between increase in SALT Score and serum levels of IL-2, IL-17A, and IL-23. (P<0.05). Th1 and Th17 pathways play a central role in the initiation and progression of AA, while Th2 pathway is suppressed in active AA. Treg pathway may be opposing Th1 and Th17 pathway and causes disease localization. The instant study lays the groundwork for understanding the pathogenesis of AA and suggests the role of implicated cytokines as potential therapeutic targets and as biomarkers of disease activity.

A Chronic Contact Eczema Impedes Migration of Antigen-Presenting Cells in Alopecia Areata

Long-lasting allergen treatment is the most efficient therapy in alopecia areata (AA). The underlying mechanism is unknown. We here asked whether treatment with a contact sensitizer influences leukocyte migration such that dendritic cell (DC) migration or the recruitment of activated T-cells towards the skin become hampered. Allergen treatment of AA mice was not accompanied by a decrease in skin-infiltrating leukocytes or draining lymph node cells (LNC). However, the distribution of leukocyte subsets was changed with a dominance of monocytes in the skin and a reduced percentage of DCs in draining nodes. Chemokine and chemokine receptor expression in skin and draining nodes was strikingly increased and LNC from untreated and allergen-treated AA mice showed high migratory activity in vitro and readily homed in draining nodes and skin after intravenous injection. However, FITC labelling of the skin and subcutaneous transfer of dye-labelled DC revealed that allergen treatment created a chemokine milieu severely hampering DC migration from the skin towards the draining node. An allergic eczema-induced reduction in DC migration and antigen transfer could well contribute to insufficient T-cell activation and the recovery of hair follicle in AA and possibly be of relevance for other skin-related autoimmune diseases.

Role of Cytotoxic T Cells in Chronic Alopecia Areata

Cytokines play a role in alopecia areata. We used immunohistochemical and in situ hybridization studies to demonstrate the persistence of pro-inflammatory as well as apoptotic mechanisms in skin biopsies from patients with chronic alopecia areata. In situ hybridization allows the visualization of the distribution of immunocompetent cells in vivo. We studied skin biopsies from 11 untreated alopecia areata patients and two normal controls. In situ hybridization was performed on frozen sections using 35S-radio-labeled riboprobes, specific for IL-1beta, IL-2, IL-6, INFgamma, and granzyme B mRNA. Immunohistochemistry was carried out using an anti-IL-1beta monoclonal antibody, and a monoclonal antibody directed against the human Fas protein. We demonstrated the presence of cells labeled with IL-1beta, IL-6, INFgamma, and granzyme B antisense probes. Similarly, cells labeled with anti-IL-1beta were found in 10 of 11 cases. The labeled cells were located in the mononuclear peri- and intrafollicular infiltrate. Cells expressing granzyme B were found in close contact with the follicle. Fas positivity was demonstrated in four of four cases at the level of the cytoplasmic membrane of the hair follicle keratinocytes. These results, based on visualizing the labeled cells, demonstrate that pro-inflammatory cytokines are produced by the mononuclear cell infiltrate in close contact with follicles in alopecia areata. Furthermore, they demonstrate for the first time that apoptotic mechanisms involving granzyme B and Fas-Fas ligand pathways may play a major role in the persistence of chronic alopecia areata.

Successful Treatment of Alopecia Areata-Like Hair Loss with the Contact Sensitizer Squaric Acid Dibutylester (SADBE) in C3H/HeJ Mice

A type of hair loss closely resembling human alopecia areata has been described in C3H/HeJ mice. In order to test the assumed analogy with human alopecia areata, we investigated the efficacy of treatment with the contact allergen squaric acid dibutylester. In 12 C3H/HeJ mice with alopecia areata an allergic contact dermatitis was induced and elicited weekly on one side of the back by topical applications of squaric acid dibutylester. Overt hair regrowth was observed only on the treated side of the back in nine of 12 mice. Histopathologic examination revealed a change in the distribution of the inflammatory infiltrate from a dense perifollicular lymphocytic infiltrate around the mid and lower regions of hair follicles in untreated skin to a uniform presence in the upper dermis in treated skin. Immunohistomorphometric studies revealed that treatment with squaric acid dibutylester increased the CD4+/CD8+ ratio from approximately 1:2 in untreated alopecia areata to 1:1 in treated alopecia areata. Additional immunohistochemical investigations showed an aberrant expression of major histocompatibility complex class I, major histocompatibility complex class II and intercellular adhesion molecule 1 on keratinocytes of the mid and lower parts of hair follicles in untreated alopecia areata. In successfully treated skin ectopic major histocompatibility complex class I and II expression was clearly reduced, whereas intercellular adhesion molecule 1 expression showed only minor changes. In conclusion, alopecia areata-like hair loss in C3H/HeJ mice responded to treatment with the contact sensitizer squaric acid dibutylester analogous to human alopecia areata. Moreover, successful treatment changes the aberrant expression of major histocompatibility complex class I and II in a way similar to that observed in human alopecia areata. These observations support the concept that alopecia areata-like hair loss in C3H/HeJ mice can be utilized as an appropriate model for the study of human alopecia areata.

IFN-gamma-induced HLA-DR but not ICAM-1 expression on cultured dermal papilla cells is downregulated by TNF-alpha.

The immune response present in untreated alopecia areata (AA) is characterized by overexpression of ICAM-1 and MHC molecules on dermal papilla cells of affected hair follicles and by a distinct cytokine pattern. After successful treatment with the potent contact allergen diphenylcyclopropenone (DCP), adhesion molecules are downregulated and a reversed pattern of cytokines is expressed. To determine which cytokines may be involved in this process we studied the expression and modulation of ICAM-1 and MHC class I and II molecules on cultured dermal papilla cells. Scalp biopsies were obtained from healthy donors and dermal papillae were isolated. The cells were treated with various cytokines and prostanoids. The surface molecules were labeled with FITC-conjugated antibodies, and the expression levels were quantified by FACScan analysis. Incubation with IFN-gamma led to a time-dependent upregulation of the surface molecules studied. IL-1 beta and TNF-alpha synergistically increased the expression of ICAM-1, but they failed to induce MHC molecules. However, both cytokines significantly reduced the IFN-gamma-induced HLA-DR expression. Pretreatment of cells with the cyclooxygenase inhibitor diclofenac, prostanoids, IL-10 or TGF-beta 1 did not alter the constitutive or IFN-gamma-elicited expression of surface molecules. A neutralizing anti-IL-1 beta-antibody did not affect any cytokine-induced changes. We conclude that with regard to surface molecules we can partly initiate in vitro the situation of AA in vivo. Moreover, our results suggest that TNF-alpha, which is markedly increased under DCP treatment, might be an effector of the therapeutic response in AA.

Cytokines and growth factors influence hair growth in vitro. Possible implications for the pathogenesis and treatment of alopecia areata.

Factors that influence the growth of the anagen hair follicle or initiate the switch to a catagen growth pattern have so far not been definitely determined, but there is increasing evidence that cytokines and growth factors play an important role during these processes. Recently we detected an aberrant in situ expression pattern of cytokines of the Th1 type (IFN gamma, IL-2) plus IL-1 beta expression in untreated alopecia areata (AA), and a switch to high levels of IL-10 TGF-beta 1 expression after successful treatment with the contact allergen diphenylcyclopropenone (DCP). Hence the question arose as to whether cytokines are able to arrest hair growth and whether IL-10 or TGF beta 1 have the capacity to antagonize this process. Using whole-organ cultures of microdissected human hair follicles we studied the effect of a panel of cytokines and growth factors on hair growth and on the gross morphology of the hair follicles in vitro. IL-2, IL-10 and IFN-gamma had no effect in this regard, whereas TGF beta 1 partially inhibited hair growth and EGF, TNF alpha and IL-1 beta completely abrogated it. EGF and TNF alpha induced the formation of a club-like hair follicle, similar to catagen morphology of the hair bulb, whereas hair follicles grown in the presence of IL-1 beta or TGF beta 1 showed no particular morphological changes. We conclude that cytokines and growth factors are pivotal regulators of hair growth at least in vitro. IL-1 is suggested as playing an important role during the pathogenesis of AA. Possible mediators of therapeutic contact dermatitis (IL-10, TGF beta 1, TNF alpha, PGE2) are, at least in vitro, not able to antagonize the IL-1 beta-triggered hair growth inhibition. Therefore, we infer that these mediators rather "modulate' the immune response in AA.

Cytokine mRNA Levels in Alopecia Areata Before and After Treatment with the Contact Allergen Diphenylcyclopropenone

Although the nature of the noxious signal and the anatomical target in alopecia areata (AA) are still unknown, it has been assumed that CD4+ T lymphocytes surrounding and infiltrating the hair bulb might trigger the hair loss. As these T lymphocytes do not promote cytotoxic activity we hypothesize that AA is triggered by cytokines. Topical immunotherapy with diphenylcyclopropenone (DCP) is at present the most effective approach. If it is true that AA results from a distinct cytokine pattern, we can hypothesize that the beneficial effect of DCP should be mediated by locally secreted cytokines during the contact allergy. Using semiquantitative reverse transcription-polymerase chain reaction with RNA extracted from scalp biopsies from patients with AA before and after successful treatment with DCP, and from healthy controls we detected a T-cell response with increased steady state mRNA levels for interferon (IFN)-gamma, interleukin (IL)-1 beta, and IL-2 in untreated AA of the totalis type. After DCP treatment, the IFN-gamma expression was reduced but still above the constitutive level found in controls, whereas mRNA expression of IL-2, IL-8, IL-10, and tumor necrosis factor-alpha was increased. Our results point towards cytokines involved in the pathogenesis in AA. A TH1 type cytokine pattern is present in untreated AA, and this is modified by cytokines secreted during DCP treatment. IL-10 has recently been described as an immunomodulator of the TH1 response and, therefore, we hypothesize that basal keratinocytes or lesional T cells secrete bioactive IL-10 after DCP application, resulting in an inhibitory effect on lesional T lymphocytes. This hypothesis would explain the effectiveness of DCP and implies the theoretical possibility of a response to topical or intralesional application of recombinant IL-10.

Abnormal Expression of Class I and Class II Major Histocompatibility Antigens in Alopecia Areata: Modulation by Topical Immunotherapy

Fifty-eight scalp biopsies were immunohistologically investigated with monoclonal antibodies against HLA-ABC, HLA-DR, and T6 antigens. The following 3 groups were compared: control biopsies obtained from healthy volunteers (n = 5) or patients with unrelated scalp diseases (n = 6); biopsies from untreated alopecia areata (AA), obtained either from untreated patients (n = 19) or from the untreated side in patients receiving unilateral treatment with the contact allergen diphencyprone (DCP) (n = 13); biopsies obtained from the treated side in patients receiving unilateral treatment with DCP (n = 13). While HLA-ABC antigens were strongly expressed by epidermal keratinocytes and the infundibular epithelium of hair follicles in all biopsies, these antigens were either not detectable or only faintly expressed on the subinfundibular epithelium and the hair matrix in the control series. By contrast, 30 out of 32 biopsies from untreated AA showed expression of HLA-ABC antigens on hair matrix epithelium, and the subinfundibular epithelium was HLA-ABC-positive in 15 out of 32 cases. In the biopsies from treated AA, HLA-ABC antigens were expressed on hair matrix epithelium in 9 out of 13 cases, and on the subinfundibular epithelium in 1 case. In the controls and untreated AA, HLA-DR expression was confined to dendritic cells in the epidermis and the follicular infundibulum. Its expression on hair matrix epithelium was found in 15 out of 32 biopsies from untreated AA and in 4 out of 13 biopsies from treated AA. In the control series, intrabulbar T6+ dendritic cells were either absent or present in low numbers. High numbers of intrabulbar T6+ cells were present in 7 out of 32 biopsies from untreated AA and in 0 out of 13 biopsies from treated AA. The data show that abnormal expression of class I major histocompatibility (MHC) antigens on hair matrix epithelium is a constant feature in AA, whereas class II MHC antigens are less frequently expressed. Topical immunotherapy with DCP, which induced expression of HLA-DR in epidermal keratinocytes in 6 out of 13 cases, reduced the abnormal expression of both HLA-ABC and -DR antigens in the epithelium of lower hair follicles in AA.

Topical immunotherapy changes the composition of the peribulbar infiltrate in alopecia areata

It has previously been shown that, in patients with untreated progressive alopecia areata (AA), the peribulbar T4/T8 ratio is about 4:1. In the present study, the immunohistochemical findings obtained in untreated AA patients were compared to those obtained in patients who had received topical immunotherapy with diphencyprone. The untreated group consisted of 5 patients with progressive AA and 5 patients with inactive AA. The treated group consisted of 5 patients with a good response to diphencyprone and 5 patients with little or no hair regrowth after treatment. In untreated patients with progressive AA, the mean peribulbar T4/T8 ratio was 4:1, whereas in untreated patients with stable AA, the ratio was 2:1. In the treated patients with a good response to diphencyprone, the mean T4/T8 ratio was 1:1, while in the patients with poor or no response to treatment, the ratio was 0.7. In conclusion, topical immunotherapy considerably alters the peribulbar T4/T8 ratio in AA. The results are consistent with, but do not prove, the concept of topical immunomodulation.

Increased number of OKT6-positive dendritic cells in the hair follicles of patients with alopecia areata.

In 6 patients with untreated alopecia areata in the progressive stage, 6 in the stationary stage, and 6 normal individuals as controls, an in situ analysis of OKT6-positive dendritic cells in hair follicles, and peribulbar and intrabulbar infiltrates was performed using the avidin-biotin-peroxidase method with monoclonal antibodies. In controls, OKT6-positive dendritic cells were distributed only in the upper portions of hair follicles and were not observed in the bulbar area, and the percentage of these cells among all epithelial cells of the hair follicles was 1.0 +/- 0.1% (mean +/- SE). In stationary-stage patients, the distribution and the percentage of positive cells were the same as those for the controls (1.1 +/- 0.1%). In the progressive stage, however, positive cells were distributed in both the upper portions of the hair follicles and the bulbar area, and the percentage of positive cells (4.9 +/- 0.3%) was significantly higher than that of controls. Staining for T, B lymphocytes and T cell subsets in the peribulbar infiltrates revealed a predominance of OKT4-positive cells (the OKT4/OKT8 ratio was from 3:1 to 4:1). This indicates that the number of OKT6-positive dendritic cells increases in the hair follicles of progressive alopecia areata and that these cells may play an important role in cooperation with T cells in the pathogenesis of alopecia areata.

Immunohistochemical analysis of T-cell subsets in the peribulbar and intrabulbar infiltrates of alopecia areata

In 11 patients with untreated alopecia areata in the progressive stage of the disease, an in situ analysis of the inflammatory infiltrate of the hair bulbs was performed by means of different monoclonal antibodies. Most of the peribulbar cells reacted with the pan T-cell antibodies OKT 3 and Lyt 3. Staining for T-cell subsets revealed that the proportion of OKT 4+ cells was about fourfold higher than that of OKT 8+ cells. Almost all of the T cells were OKIa1+, indicating that they were in an activated state. In four of the 11 cases, both subsets of T lymphocytes were also found to infiltrate the hair matrix itself. These results would appear to be consistent with the assumption that alopecia areata is caused by a T cell mediated autoimmune mechanism.