The Contributory Roles of Th17 Lymphocyte and Cytotoxic T Lymphocyte at the Hair Bulge Region as Well as the Hair Bulb Area in the Chronic Alopecia Areata Patients

Abstract

Background: Alopecia areata (AA) is a T cell-mediated autoimmune disease that targets hair follicles and interrupts hair regrowth. The microenvironment of the effector T cells and their related cytokines may affect immunopathogenesis around the hair bulb/bulge.

Objective: To determine the contributory roles of the effector T cell subsets and related cytokines to the pathogenesis of AA.

Methods: We investigated the correlation between histopathological grades and four clinical prognostic factors in 331 patients with AA, and analyzed the topography of T cell infiltrates and related cytokines around the hair bulb/bulge according to histopathological grades through immunohistochemical and double immunofluorescence studies on a subset of AA specimens.

Results: First, the groups with more severe histopathological grades were associated with earlier onset, longer duration, more hair loss, as well as poorer therapeutic outcomes. Second, the pattern of CD4 and CD8 expression around the hair bulb/bulge varied by histopathological grade, with staining density decreasing in the following order: type 1>type 2>type 3. In addition, interferon-γ and transforming growth factor-β1 expression appeared denser in the peribulbar area. Interestingly, the denser CCR6⁺ cells (Th17 cells) showed more infiltration than CCR5⁺ cells (Th1 cells) around the hair bulb/bulge as histopathological grade worsened.

Conclusion: The insidious destruction of bulge stem cells and hair bulb matrix stem cells results in more severe hair loss in patients with chronic AA, which is mediated by Th17 lymphocyte and cytotoxic T lymphocyte infiltration. Furthermore, Th17 lymphocytes may play an even more important role than cytotoxic T cells in the development of AA.

Keywords: Chronic alopecia areata; Cytotoxic T lymphocyte; Th17 cells.

Fig. 1. Immunnohistochemistries for CD4⁺ and CD8⁺ lymphocytes. CD4 positive cells infiltrated mostly in peribulbar area. CD8⁺ cells infiltrated mostly in intrabulbar area. The severer histopathologic gradings were associated with the denser infiltrations of mononuclear cell (A~C: CD4, ×100), (D~F, CD8, ×100).

Fig. 2. CD4 and CD8 immunohistochemistries for types 1, 2, and 3 hair follicles in alopecia areata (AA) patients: (A) and (D), type 3 AA hair follicles, (B) and (E); type 2 AA hair follicles, (C) and (F); type 1 AA hair follicles. At hair bulb (Bb) areas, CD4⁺ T lymphocytes infiltrated perifollicularly and CD8⁺ T lymphocytes infiltrated intrafollicularly, Interestingly, the denser CD4⁺ and CD8⁺ T lymphocytes infiltrated around hair bulge (Bg) regions as the histopathologic gradings were worsened.

Fig. 3. Expression of CCR6 and CCL20 within type 1 hair bulbs (B~D), type 2 hair bulbs (F~H), and type 3 hair bulbs (J~L). A representative finding of double immunofluoroscence showed immunoreactive cells for CCR6 (B, F, J; green) and CCL20 (C, G, K; red) withco-localization study of CCR6 and CCL20 (D, H, L). Same samples for H&E stain were also shown from type 1 to type 3 (A, E ,I). The denser CCR6 and CCL20 were expressed in parallel with the severer histopathologic gradings (H&E: A, E, I, ×100; double immunofluorescence: B~D, F~H, J~L, ×100).

Fig. 4. Expression of CCR6 and CCR5 within type 1 hair bulbs (B~D), type 2 hair bulbs (F~H), and type 3 hair bulbs (J~L). A representative finding of double immunofluorescences immunoreactive cells for CCR6 (B, F, J; green) and CCR5 (C, G, K; red) with colocalization study of CCR6 and CCR5 (D, H, L). H&E stained sectionswere also shown from type 1 to type 3 (A, E, I). The denser CCR6 and CCR5 were expressed in parallel with the severer histopathologic gradings (H&E: A, E, I, ×100; double immunofluorescence, B~D, F~H, J~L, ×100).

Fig. 5. Expression of CCR6 and CCL20 within type 1 hair bulges (A~C), type 2 hair bulges (D~F), and type 3 hair bulges (G~I).

Fig. 6. Immunnohistochemistry for interferon (IFN)-γ. IFN-γ wasexpressed in the inflammatory cells around the hair bulb, and the severer histopathologic grading was associated with the denser immunoreactive cells for IFN-γ (A~C, ×100; D~F, ×200).

Fig. 7. Immunnohistochemistry for transforming growth factor (TGF)-β1. Expression of TGF-β1 was detected in the outer root sheath keratinocyte, regressing epithelial strand, dermal sheath and dermal sheath cells, and its expression was correlated with histopathologic grading (A~C, ×100; D~F, ×200).

Fig. 8. Immunnohistochemistry for transforming growth factor (TGF)-β2. TGF-β2 was expressed in the dermal papilla area, and less expression was correlated with the severer histopathologic gradings (A~C, ×100; D~F, ×200).