IL-31 receptor alpha expression in epidermal keratinocytes is modulated by cell differentiation and interferon gamma.

TO THE EDITOR Recently, IL-31 has been identified as a short-chain 4-helix bundle cytokine that is expressed by activated CD4+ T cells, preferentially by T cells skewed toward a T helper type 2 TH2-type phenotype (Dillon et al., 2004). IL-31 signals through a heteromeric receptor complex composed of the IL-31 receptor alpha (IL-31Rα) and the oncostatin M receptor beta (Dillon et al., 2004). The IL-31Rα was originally identified as gp130-like monocyte receptor (Ghilardi et al., 2002) and gp130-like receptor (Diveu et al., 2003, Dreuw et al., 2004) and shows 28% homology to gp130, the common signaling receptor subunit of the family of IL-6-type cytokines. Expressions of IL-31Rα and oncostatin M receptor beta mRNA have been shown to be induced in activated monocytes, whereas tissues of the skin, testis, thymus, and trachea, as well as intestinal epithelial cells and dorsal root ganglia express mRNA for both receptors constitutively (Dillon et al., 2004; Bando et al., 2006; Sonkoly et al., 2006; Dambacher et al., 2007). Engagement of the receptor complex resulted in activation of Jak1, and to a minor extent of Jak2, as well as STAT1, STAT3, STAT5, and MAPK and PI3K signaling pathways in glioblastoma and melanoma tumor cells and lung epithelial cells (Diveu et al., 2004; Chattopadhyay et al., 2007, Dambacher et al., 2007). So far, biological functions of this previously unknown cytokine were mainly analyzed in skin diseases such as atopic dermatitis (AD) or allergic contact dermatitis, in which increased expression rates of IL-31 were detected (Neis et al., 2006, Bilsborough et al., 2006, Sonkoly et al., 2006). In vivo, Staphylococcal superantigen strongly induced IL-31 expression in PBMCs obtained from patients with AD (Sonkoly et al., 2006). In psoriatic plaques, expression of IL-31 was absent, confirming an involvement of IL-31 in TH2-mediated skin diseases. Likewise, in NC/Nga mice, an experimental animal model for AD, a good correlation between the scratching counts and expression of IL-31 mRNA was demonstrated (Takaoka et al., 2005, 2006), and transgenic mice overexpressing IL-31 develop a skin disorder characterized by severe pruritus, alopecia, and skin lesions (Dillon et al., 2004). Although an enhanced expression of IL-31Rα has been described in inflammatory skin diseases, the influence of cell differentiation on the expression of the IL-31Rα in skin cells was largely unknown. We therefore analyzed the effects of the cellular differentiation state on epidermal mRNA and protein levels of IL-31Rα under culture conditions when epidermal cultures exhibit predominantly basal, spinous, and granular cell phenotypes (Du et al., 2006). NHEKs were obtained from foreskin specimen by dispase separation (BD Biosciences) of the epidemal sheet from the dermis and subsequent trypsin digestion (Cambrex, Walkersville, MD) of the epidermis (Baron et al., 2005). All studies were approved by the ethical committee of the University Hospital, RWTH, Aachen. Participants gave their written informed consent, and the study was conducted according to the Declaration of Helsinki Principles. The differentiated keratinocyte phenotype was defined by cellular morphology and expression of differentiation-specific gene markers. Cytokeratin 10 mRNA (KRT 10) expression, a specific marker of the basal to spinous transition in vivo, was upregulated during the first week of in vitro differentiation, achieving maximal levels at days 5 and 10 (Figure 1a). The upregulation of loricrin mRNA, a specific marker of the spinous to granular transition in vivo, required 2 weeks of cultivation of keratinocytes in differentiation medium (1.4 mM Ca2+) (Figure 1b). Interestingly, IL-31Rα mRNA completely disappeared during the process of differentiation. Already at the transition from basal to spinous phenotype (d5), hardly any mRNA was detectable by quantitative reverse transcription-PCR (Figure 1c). Expression of oncostatin M receptor beta mRNA was decreased on day 5 but not significantly altered during the late phases of keratinocyte differentiation (Figure 1d). Using an IL-31Rα-specific antibody (R&D Systems, Wiesbaden-Nordenstadt), we confirmed a strong downregulation of the receptor at the protein level when differentiation of normal human epidermal keratinocytes (NHEK) was induced by culturing the cells in differentiation medium containing 1.4 mM Ca2+ for more than 3 days (Figure 2a). Protein expression was not traceable in terminal differentiated keratinocytes (day 15) resembling granular cell phenotype. Additionally, we did not find IL-31Rα expression in dermal fibroblasts (Figure 2a, lane 8), which is in agreement with the findings by Chattopadhyay et al. (2007) that fibroblastic cells did not respond to IL-31 treatment (Chattopadhyay et al., 2007). Monocytes induced with 1000 U ml−1 IFN-γ for 48 h revealed a strong upregulation of IL-31Rα expression (Figure 2a, lane 7) (Dillon et al., 2004). Stimulation of proliferating NHEK with IFN-γ led to an upregulation of IL-31Rα mRNA (Figure 1e) and protein expression (Figure 2b, lane 4). Stimulation of NHEK in early phases of differentiation (d6, d8; Figure 1f) with IFN-γ also revealed an upregulation of IL-31Rα expression. In the late phases of differentiation (d12, d15, data not shown), IFN-γ treatment of NHEK had no effect of IL-31Rα expression. In inflamed skin, keratinocytes in the early stages of differentiation might therefore be more responsive to IL-31. Challenge with IL-1α, tumor necrosis factor-α (TNF-α) and a mixture of pro-inflammatory cytokines (TNF-α, IL-1α, and IL-6; Tjabringa et al., 2007) did not alter IL-31Rα expression in proliferating or differentiated NHEK (Figure 1e and f, 2b, lane 5). Loss of receptor expression in late-stage keratinocytes consequently led to a loss of cytokine responsiveness; although proliferating keratinocytes respond to IL-31 treatment with increased tyrosine phosphorylation of STAT3, we could not detect any STAT activation in differentiated keratinocytes (Figure 2c). In contrast, stimulation with OSM resulted in STAT3 tyrosine phosphorylation irrespective of the differentiation state, which indicates that differentiated keratinocytes are not unresponsive to cytokine treatment, in general (Figure 2c). Primary keratinocytes are known to express both IL-31 receptor components and respond to IL-31 stimulation with the production of chemokines such as thymus and activation-regulated chemokine (TARC/CCL17) or macrophage-derived chemokine (MDC/CCL22; Dillon et al., 2004). Using immunohistology, Bilsborough et al. (2006) have demonstrated that levels of IL-31Rα expressed on keratinocytes from AD skin biopsy specimens were consistently higher than the levels observed in skin biopsy specimen from healthy control subjects. These data correspond to our findings, as proliferating keratinocytes show higher levels of IL-31Rα and remain sensitive to IL-31 stimulation, whereas differentiated cells become unresponsive (Figure 1c, 2a and c). Recently, a prominent involvement of activated Th1-subset of T-cells and increased expression of receptor of IFN-γ on keratinocytes has been detected in acute skin lesions of AD (Lugović et al., 2005). In our studies, INF-γ had a strong stimulatory influence on the expression of IL-31Rα in proliferating NEHK (Figure 1e, 2b). Additional GeneChip Human Exon 1.0 ST array analysis of proliferating and differentiated NHEK (data not shown) revealed a downregulation of IFN-γ receptor 2 in differentiated keratinocytes, which may correlate with the downregulation of the IL-31Rα detected in these cells (Figure 1c, 2a). Broxmeyer et al. (2007) demonstrated a survival-enhancing effect of IL-31 on murine hematogenic progenitor cells in vitro but no modulation of proliferation. So far, IL-31 has been shown to have a strong growth inhibitory activity on epithelial cells (Chattopadhyay et al., 2007, Dambacher et al., 2007). Chattopadhyay et al. (2007) delineated that IL-31 was highly effective in suppressing proliferation by altering expression of cell-cycle proteins, including upregulation of p27Kip1 and downregulation of cyclin B1, CDC2, CDK2, MCM4, and the retinoblastoma protein. Therefore, cells seem to be arrested in G0 phase without induction of apoptosis. Whether IL-31 might be involved in the molecular switch from proliferation to differentiation needs to be determined. In conclusion, in vitro studies analyzing the effects of the previously unknown cytokine IL-31 on epidermal keratinocytes should consider the variation of IL-31Rα expression, which depends on the status of cellular differentiation and the influence of pro-inflammatory cytokines. Further clinical studies and highly specific mAbs are necessary to assess the impact of these findings in vivo. The authors state no conflict of interest. These studies were supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 542, TP C11)