IL-6 Negatively Regulates IL-22R α Expression on Epidermal Keratinocytes: Implications for Irritant Contact Dermatitis

Abstract

Irritant Contact Dermatitis (ICD) is characterized by epidermal hyperplasia and inflammatory cytokine release. IL-6 has been shown to be involved in the pathogenesis of ICD; however, the involvement of the IL-22/IL-22Rα axis and its relation to IL-6 in the inflammatory response following irritant exposure are unknown. Using a chemical model of ICD, it was observed that mice with a keratinocyte-specific knockout of IL-6Rα (IL-6Rα ^Δker) presented with increased inflammation and IL-22Rα and IL-22 protein expression relative to WT following irritant exposure, indicating that IL-6Rα deficiency in epidermal keratinocytes leads to the upregulation of IL-22Rα and its ligand during ICD. Furthermore, it was shown that IL-6 negatively regulates the expression of IL-22Rα on epidermal keratinocytes. This effect is functional as the effects of IL-22 on keratinocyte proliferation and differentiation were markedly reduced when keratinocytes were pretreated with IL-6 prior to IL-22 treatment. These results show that IL-6 modulates the IL-22/IL-22Rα axis in the skin and suggest that this occurrence may be associated with the increased epidermal hyperplasia and exacerbated inflammatory response observed in IL-6Rα ^Δker mice during ICD.

Figure 1

IL-6Rα^Δker mice present with increased epidermal hyperplasia following irritant exposure. Loss of the IL-6Rα in keratinocytes promotes epidermal hyperplasia during irritant contact dermatitis. WT and IL-6Rα^Δker mice were exposed to BKC or control for seven (7) consecutive days to induce ICD. 24 hours after irritant exposure, 8 mm biopsies of lesional skin were collected and embedded in O.C.T. compound for histological analysis. Skin samples were cross-sectioned and then hematoxylin and eosin (H&E) stained. Representative H&E stains from WT (a, b) and IL-6Rα^Δker (c, d) are shown. Quantification of epidermal thickness (e) and cells per field (f) as determined by ImageJ (NIH) is presented. Data are mean ± SD. ^∗Significantly different from WT (p ≤ 0.05, n = 15 mice/treatment/genotype).

Figure 2

IL-6Rα^Δker mice express higher levels of IL-22Rα and IL-22 in lesional skin. Irritants induce higher expression of IL-22 and IL-22Rα in mice with a keratinocyte-specific knockout of IL-6Rα, IL-6Rα^Δker, and WT mice were exposed to BKC or control for 7 consecutive days. Lesional skin was harvested from each genotype and IL-22 protein expression was determined by Luminex immunoassay (a). Immunohistochemical analysis of lesional skin from irritant-exposed mice, stained for the expression of IL-22Rα (green), and nuclear staining DAPI (blue). Representative images from WT (b, c) and IL-6Rα^Δker (d, e). Quantification of IL-22Rα expression as determined by ImageJ (NIH) is presented (f). Data are mean ± SD. ^∗Significantly different from WT (p ≤ 0.05, n = 15 mice/treatment/genotype).

Figure 3

IL-6 negatively regulates IL-22Rα expression on epidermal keratinocytes. Primary keratinocytes from IL-6KO mice were treated with rmIL-6 for 4/24 hours (mRNA/protein expression) at the indicated concentrations. The expression of IL-22Rα mRNA was analyzed and normalized to 28S ribosomal RNA as control (a). IL-6KO keratinocytes were grown to confluency on multichamber slides. Immunohistochemical analysis of keratinocyte culture stained for the expression of IL-22Rα (green), and nuclear staining DAPI (blue). Representative fluorescent images are shown at 20x (b–e) and 40x (f–i). Quantification of IL-22Rα expression as determined by ImageJ (NIH) is presented (j and k). Data are mean ± SD. ^∗Significantly different from 0 ng/ml rmIL-6 (p ≤ 0.05, n = 3 separate experiments).

Figure 4

IL-6 reduces the effect of IL-22 on keratinocyte differentiation and proliferation. Primary keratinocytes were pretreated with rmIL-6 for 24 hours followed by exposure to 20 ng/ml rmIL-22 for 24 hours (a–f and g–j) or 4 hours (k and l). Immunohistochemical analysis of IL-6KO keratinocyte expression of KRT1 (a–f) or Ki67 (g–j) (green) protein and nuclear staining with DAPI (blue). Representative images are shown. KRT1 expression (k) and Ki67 (l) mRNA expression were analyzed by RT-PCR. Keratinocytes were exposed to rmIL-6 alone in a dose-dependent manner for 4 hours, and KRT1 (m) and Ki67 (n) mRNA were analyzed by RT-PCR. Data are mean ± SD. ^∗Significantly different from 0 ng/ml rmIL-6 (p ≤ 0.05, n = 4 separate experiments).