Production of IL-31 in CD45RO+CLA+H4R+ T Cells in Atopic Dermatitis

Abstract

IL-31 is involved in pruritus in atopic dermatitis (AD) and the pathogenesis of AD. However, the mechanism of IL-31 production is not fully understood. We sought to investigate the association between CD45RO⁺CLA⁺H4R⁺ T cells and IL-31 production. Immunofluorescence studies were performed retrospectively on punch-biopsy specimens from five people with AD and three healthy controls. In addition, blood samples were collected prospectively from eight patients with AD and eight healthy controls for sorting CD45RO⁺CLA⁺H4R⁺ T cells. There was no overlap of patients between the biopsy group and the blood sampling group. Sorted cells were stimulated with 4-methylhistamine (4MH), and the level of IL-31 was measured by an enzyme-linked immunosorbent assay. Immunofluorescence showed co-localization of H4R and IL-31 in lesional AD skin but not in normal skin of healthy controls. The proportion of CLA⁺H4R⁺ T cells among CD3⁺CD45RO⁺ lymphocytes was 18.3 ± 6.2% in patients with AD and 11.2 ± 7.6% in healthy controls. In the AD group, production of IL-31 by CD45RO⁺CLA⁺H4R⁺ T cells increased from 32.4 ± 13.3 pg/mL to 47.5 ± 18.7 pg/mL by 4MH stimulation after 24 h (p < 0.001). However, in the control group, production of IL-31 was 20.1 ± 10.6 pg/mL without and 22.1 ± 9.3 pg/mL with 4MH stimulation (p > 0.05). According to our study, CD45RO⁺CLA⁺H4R⁺ T cells are an important source of IL-31 in AD, and may be a target for treatment of IL-31-induced pruritus.

Keywords: Interleukin-31; atopic dermatitis; histamine-4-receptor.

Figure 1

Co-localization of IL-31 and H4R in AD and healthy controls. (A) Overlay (yellow) of IL-31⁺ (green) and H4R⁺ (red) in AD samples and healthy controls. DAPI (blue) was used to counterstain nuclei. Original magnification × 200, scale bar = 100 μm for control and reticular dermis of AD. Original magnification × 100, scale bar = 200 μm for papillary dermis of AD. (B) IL-31⁺ and H4R⁺ co-localized cell counts in healthy controls and patients with AD. High-power field, × 200. Error bars indicate SEMs.

Figure 2

Result of CD45RO⁺CLA⁺H4R⁺ T cells in AD and healthy controls. The FACS results of CD45RO⁺CLA⁺H4R⁺ T cells in (A) healthy controls and (B) patients with AD. (C) Proportion of CLA⁺H4R⁺ T cells in CD3⁺CD45RO⁺ T cells; 9.1 ± 5.6% in patients with AD and 4.8 ± 3.8% in healthy control. AD, atopic dermatitis. FACS, fluorescence-activated cell sorting.

Figure 3

Simultaneous expression of IL-31 and H4R in sorted CD45RO⁺CLA⁺H4R⁺ T cells of AD patients with 4-methylhistamine (4MH) stimulation. Isolated CD45RO⁺CLA⁺H4R⁺ T cells from healthy controls and AD samples were cultured and stimulated with or without 4MH at 10 μM. Immunofluorescence images of IL-31 (green) and H4R (red) in sorted CD45RO⁺CLA⁺H4R⁺ T cells were observed with original magnification × 400, scale bar = 50 μm.

Figure 4

The production of IL-31 in CD45RO⁺CLA⁺H4R⁺ T cells and CD45RO⁺CLA⁺H4R⁻ T cells. CD45RO⁺CLA⁺H4R⁻ T cells and CD45RO⁺CLA⁺H4R⁺ T cells were isolated from blood samples acquired from healthy controls and patients with AD. These sorted cells were cultured at a density of 1.5 × 10⁴ cells/well and stimulated with or without 4MH at 10 μM. After stimulation for 24 h, supernatant harvested from cell medium was evaluated by ELISA for level of IL-31. * p < 0.05, *** p < 0.001.