IL-27 Regulates IL-18 binding protein in skin resident cells

Abstract

IL-18 is an important mediator involved in chronic inflammatory conditions such as cutaneous lupus erythematosus, psoriasis and chronic eczema. An imbalance between IL-18 and its endogenous antagonist IL-18 binding protein (BP) may account for increased IL-18 activity. IL-27 is a cytokine with dual function displaying pro- and anti-inflammatory properties. Here we provide evidence for a yet not described anti-inflammatory mode of action on skin resident cells. Human keratinocytes and surprisingly also fibroblasts (which do not produce any IL-18) show a robust, dose-dependent and highly inducible mRNA expression and secretion of IL-18BP upon IL-27 stimulation. Other IL-12 family members failed to induce IL-18BP. The production of IL-18BP peaked between 48-72 h after stimulation and was sustained for up to 96 h. Investigation of the signalling pathway showed that IL-27 activates STAT1 in human keratinocytes and that a proximal GAS site at the IL-18BP promoter is of importance for the functional activity of IL-27. The data are in support of a significant anti-inflammatory effect of IL-27 on skin resident cells. An important novel property of IL-27 in skin pathobiology may be to counter-regulate IL-18 activities by acting on keratinocytes and importantly also on dermal fibroblasts.

Figure 1. IL-27 dose-depenently induces IL-18BP secretion in human keratinocytes.

Human primary keratinocytes (a, b, c) or HaCat (d) were stimulated for 48 h. Cell free supernatant was harvested and IL-18BP content was determined by Elisa. Independent experiments were performed. (a) n = 4 different experiments and donors; (b) n = 7 different experiments and donors; (c) n = 3 different experiments and donors; (d) n = 3. Mean and SEM are depicted. ns = non stimulated, HPK = human primary keratinocytes.

Figure 2. Human fibroblasts are very responsive to IL-27 stimulation.

Stimulation of primary human skin fibroblasts was performed for 48 h and cell-free supernatants were analysed for IL-18BP by Elisa. Results are given as mean and SEM. (a) n = 7 (b) n = 4, ns = non stimulated.

Figure 3. Time course of IL-18BP release by IL-27 stimulated skin cells.

Fibroblasts (a) and HaCat cells (b) were cultured for up to 96 h after initial stimulation with IL-27 (50 ng/ml). Supernatants were collected at the indicated time points. Levels of IL-18BP for non stimulated cells were below the detection limit of the ELISA. n = 4 (a, b).

Figure 4. IL-18BP mRNA induction by IL-27.

Fibroblasts (a,c) and HaCat cells (b) were stimulated with IL-27 (50 ng/ml) for 5 h or overnight (16 h). qRT-PCR was performed and results were normalised to the expression of the housekeeping gene U6. The result obtained for non stimulated cells (5 h; not depicted) was used as “calibrator” (defined as 1). Stability of the IL-18BP and IL-8 mRNA stability was analysed in IL-27 (50 ng/ml) stimulated cells using actinomycin D (AD). Results were normalised to the expression of the housekeeping gene U6snRNA and the value obtained for cells not treated with AD ( =  no AD) was used as “calibrator” and defined as 1. (a) n = 3, (b) n = 2, (c) one out of 2 independent experiments is depicted. ns = non stimulated.

Figure 5. IL-27 induced IL-18BP activation pathway in HaCat cells.

(a) HaCat cells were stimulated for 30 min with IL-27 (100 ng/ml), IFNγ (20 ng/ml) or used as non stimulated control, lysed and the obtained nuclear extract analysed by western blot using antibodies specific for total STAT1 and pSTAT1-Y701. One representative of three independently performed experiments is shown. (b) HaCat cells were stimulated for 24 h with with IL-27 (50 ng/ml), IFNγ (20 ng/ml) or used as unstimulated control and mRNA expression of IL-18BP was determined by qRT-PCR. IL-18BP mRNA was normalized to that of GAPDH and is shown as mean fold induction compared to unstimulated control ± S.D. (n = 6). (c) HaCat cells were transfected with the indicated IL-18BP promoter constructs. After 24 h, cells were kept as non-stimulated control or stimulated with IL-27 (100 ng/ml). After another 24 h, cells were harvested and luciferase assays were performed. Data are expressed as mean fold-luciferase induction ± SD (compared to the non-stimulated control transfected with the same plasmid) obtained from 4 independent experiments. *p<0.05 and **p<0.01 compared to non stimulated control of the respective plasmid; ^#p<0.05 compared pGL3-BPwt under the influence of IL-27, $$p<0.01 compared to pGL3-BPmt/dist under the influence of IL-27.