IL-21R mRNA is overexpressed in systemic sclerosis and contributes to the epidermal/dermal cross-talk

Objectives

IL-21R is a class I cytokine receptor associated with the common γ-chain. In mice, IL-21R was detected in fibrotic lungs but not in healthy lungs, suggesting a possible role for IL-21R in fibrotic disorders.

Methods

Skin biopsies were obtained from 12 patients with systemic sclerosis (SSc) and 11 healthy controls. Total RNA was isolated from these tissues, reverse transcribed, and IL-21/IL-21R mRNA was quantified using real-time PCR. The expression patterns of IL-21/IL-21R were analyzed by in situ hybridization. Stimulation experiments were performed with cultured dermal fibroblasts from SSc patients and healthy controls as well as keratinocytes using IL-1β, platelet derived growth factor-BB (PDGF-BB), monocyte chemoattractant protein-1 (MCP-1), transforming growth factor (TGF)-β and IL-21. Human skin biopsies from SSc patients were transplanted onto SCID mice. After 60 days, the expression of IL-21R was determined by in situ hybridization. Cultured keratinocytes were stimulated with IL-21 and assessed for differential expression compared with nonstimulated controls using a human Atlas cDNA expression array (Clontech, Palo Alto, CA, USA).

Results

IL-21R was detected in all biopsies from SSc patients and controls with a 4.7-fold increase in SSc samples (threshold cycle: SSc patients, 17.9 ± 1.1; healthy, 20.1 ± 1.4). In situ hybridization showed an upregulation of IL-21R in the epidermis of SSc patients, whereas no signal was detected in healthy skin specimens. These results were confirmed in vitro, in that cultured keratinocytes expressed significant levels of IL-21R, whereas no signal was found in fibroblasts. Interestingly, mRNA for IL-21, the proposed ligand for IL-21R, was not detected by real-time PCR and in situ hybridization. Various concentrations of IL-1β, PDGF-BB, MCP-1, TGF-β and IL-21 did not stimulate the expression of IL-21R in cultured keratinocytes and dermal fibroblasts. In the SCID mouse transplantation model, the overexpression of IL-21R in SSc keratinocytes remained unchanged as long as 60 days after transplantation, whereas no signals for IL-21R were detected in fibroblasts. Differentially expressed genes confirmed by SYBR Green real-time PCR after stimulation with IL-21 included vascular endothelial growth factor, vascular endothelial growth factor-C and integrin a6.

Conclusion

This is the first human disease-related report regarding IL-21R in vivo. These results indicate that, in addition to fibroblasts and endothelial cells, the expression pattern of keratinocytes is altered in patients with SSc. Interestingly, the expression of IL-21R appears to be independent of inflammatory cytokines in vitro and independent of both humoral and cellular factors circulating in SSc patients, because the expression of IL-21R mRNA in human epidermal keratinocytes did not change after implantation onto SCID mice.