Morphological Alterations and Increased S100B Expression in Epidermal Langerhans Cells Detected in Skin from Patients with Progressive Vitiligo

Abstract

The role of Langerhans cells (LCs) in vitiligo pathogenesis remains unclear, with published studies reporting contradictory results regarding the quantity of LCs and no data on the features of LCs in vitiligo. Here, we aimed to analyze the presence, density, and morphological features of LCs in the epidermis of patients with vitiligo. Skin biopsies were stained for LCs using anti-CD1a/anti-langerin antibodies and analyzed by immunocytochemistry with light and electron microscopy. Compared with healthy controls, we detected significantly increased numbers of epidermal LCs in lesional skin from vitiligo in the progressive state. These LCs exhibited striking morphological alterations, including an elevated number of dendrites, with increased length and more branches than dendrites from controls. Ultrastructure examination via immuno-electron microscopy revealed markedly reduced Birbeck granules (BGs) and shorter BG rods in LCs from progressive vitiligo, with higher expression of langerin. Additionally, expression of S100B, the activity biomarker of vitiligo, was increased in these LCs. This work provides new insight on the cellular composition of LCs in vitiliginous skin, revealing altered morphology and increased LC numbers, with elevated S100B expression. Our data suggest LCs might play a critical role in vitiligo pathogenesis and thus may represent a novel therapeutic target for this disease.

Keywords: Birbeck granule; Langerhans cells; S100B; leukoderma; vitiligo.

Figure 1

Epidermal Langerhans cells (LCs) are increased in lesional skin from progressive vitiligo relative to stable vitiligo and normal skin. Skin sections from healthy controls (n = 5), non-segmental vitiligo in the progressive state (Progressive Vitiligo, n = 8), and non-segmental vitiligo in the stable state (Stable Vitiligo, n = 10) were stained with anti-CD1a (green) and anti-Melan-A (red) antibodies. Nuclei were stained in blue with Hoechst 33342. Representative images are shown, and the corresponding bright-field microscopy images are displayed in the upper panels. Scale bar: 100 μm.

Figure 2

Increased number of LCs, dendrites per LC, and dendrite length in lesional skin from progressive vitiligo and progressive rhododendrol-induced leukoderma (RDIL). (a) Skin sections from healthy controls (n = 5), non-segmental vitiligo in the progressive state (Progressive Vitiligo, n = 8), and RDIL in the progressive state (RDIL, n = 13) were stained with anti-CD1a antibodies (green). Nuclei were stained in blue with Hoechst 33342. Representative images are shown, with higher-magnification images corresponding to the area surrounded by the red rectangles in left panels shown at right. Scale bar: 50 μm. Quantification of LCs (percentage of total cells in epidermis) (b), number of dendrites per LC (c), and length of dendrites (d) in LCs. Data in (b–d) are shown as the means ± standard deviation (SD). *** p < 0.01.

Figure 3

Immuno-transmission electron microscopy (TEM) reveals altered Birbeck granule (BG) morphology in epidermal LCs from progressive vitiligo. Skin sections from healthy controls (n = 3) and non-segmental vitiligo in a progressive state (Progressive Vitiligo, n = 3) were stained with anti-langerin antibodies, and immuno-TEM was performed. (a) Representative TEM images are shown; areas framed in white rectangles are enlarged in right panels. Small black dots represent 30–50 nm sized colloidal gold (enhanced from 1.4 nm nanogold) particles labeling langerin. Scale bar: 1 μm. (b) Representative high-power TEM images of BGs in healthy controls and progressive vitiligo. Scale bar: 200 nm. Quantification of BGs per LC (c), rod length of BGs (d), and langerin colloids per LC (e) in progressive vitiligo and healthy controls. Data in (c–e) are shown as means ± SD. *** p < 0.01.

Figure 4

Increased S100B expression level in LCs from the skin of subjects with progressive vitiligo. (a) Skin sections from healthy control (n = 5), non-segmental vitiligo in a progressive state (Progressive Vitiligo, n = 8), and non-segmental vitiligo in a stable state (Stable Vitiligo, n = 10) were stained with anti-Melan-A (white), anti-CD1a (green), and anti-S100B (red) antibodies. Nuclei were stained in blue with Hoechst 33342. Representative images are shown, and the corresponding bright-field microscopy images are displayed in the upper panels. Scale bar: 100 μm. (b) Quantification of LCs (percentage of total cells in epidermis); (c) quantification of S100B expression level in LCs (relative fluorescent intensity compared with healthy control). Data in (b,c) are shown as means ± SD. *** p < 0.01, n.s., no statistical significance.